Anti-lamp1 antibodies and antibody drug conjugates, and uses thereof

ABSTRACT

Antibodies are provided which specifically bind human and  Macaca fascicularis  lysosomal-associated membrane protein 1 (LAMP1) proteins and immunoconjugates comprising said antibodies conjugated or linked to a growth inhibitory agent. Pharmaceutical compositions comprising antibodies or immunoconjugates of the invention and use of the antibodies or immunoconjugates for the treatment of cancer are also provided, as well as LAMP1 antibodies, isolated nucleic acids, vectors and host cells comprising a sequence encoding said antibodies and the use of said antibody as a diagnostic tool. The application further provides for the detection of LAMP1 gene amplification or gain in cancer cells leading to the determination if patients with cancer are likely to respond to anti-LAMP1 therapy. Therefore, it is proposed an in vitro method of selecting patients with cancer which comprises determining, in a biological sample of a patient with cancer which includes cancer cells, if said patient harbors a LAMP1 gene copy number gain; and selecting the patient based on the presence of LAMP1 gene copy number gain. Anti-LAMP1 therapeutic agent for use for treating cancer in a patient harboring LAMP1 gene copy number gain in cancer cells is further provided.

This application is a divisional of U.S. patent application Ser. No.14/751,598, filed Jun. 26, 2015, which is a continuation ofInternational Application No. PCT/EP2013/078017, filed Dec. 26, 2013,which claims priority to European Patent Application No. 12306694.6,filed Dec. 27, 2012, and to European Patent Application No. 12306691.2,filed Dec. 27, 2012, all of which are incorporated herein by referencein their entirety.

BACKGROUND

Antibodies are provided which specifically bind human and Macacafascicularis lysosomal-associated membrane protein 1 (LAMP1) proteinsand immunoconjugates comprising said antibodies conjugated or linked toa growth inhibitory agent. Pharmaceutical compositions comprisingantibodies or immunoconjugates of the invention and use of theantibodies or immunoconjugates for the treatment of cancer are alsoprovided, as well as LAMP1 antibodies, isolated nucleic acids, vectorsand host cells comprising a sequence encoding said antibodies and theuse of said antibody as a diagnostic tool. The application furtherprovides for the detection of LAMP1 gene amplification or gain in cancercells leading to the determination if patients with cancer are likely torespond to anti-LAMP1 therapy. Therefore, it is proposed an in vitromethod of selecting patients with cancer which comprises determining, ina biological sample of a patient with cancer which includes cancercells, if said patient harbors a LAMP1 gene copy number gain; andselecting the patient based on the presence of LAMP1 gene copy numbergain. Anti-LAMP1 therapeutic agent for use for treating cancer in apatient harboring LAMP1 gene copy number gain in cancer cells is furtherprovided.

Lysosome-associated membrane protein 1 (LAMP1), also known as CD107antigen-like family member A (CD107a), is a single-pass type I membraneprotein, which belongs to the LAMP family. LAMP2 is the closest memberof the family and both proteins are the most abundant glycoproteinswithin the lysosomal membrane (Sawada, R. et al., 1993, J Biol Chem 268:12675-12681).

Although encoded by separate genes, with LAMP1 located on chromosome13q34 and LAMP2 on Xq24-25, the proteins are similar in their primarystructure, with ˜36% sequence homology (Mattei, M. G. et al., 1990, JBiol Chem 265:7548-7551). LAMP1 and LAMP2 consist of a polypeptide coreof approximately 40 kDa; they are both anchored via their C-terminus tothe lysosomal membrane and expose the largest part, extensivelyglycosylated, to the lumenal side of lysosomes. Both proteins are amongthe most heavily glycosylated of cellular proteins with ˜50% of theirmass as carbohydrates and these glycosylations seem to be the key formaintaining lysosome acidity and protecting the lysosomal membranes fromautodigestion. However, the full biological function of these two highlyglycosylated proteins in particular LAMP1 still needs to be elucidated(Fukuda, M., 1991, J Biol Chem, 266:21327-21330; Winchester, B., 2001,European Journal of Paediatry Neurology, 5:11-19; Gasnier, B., 2009Biochimica et Biophysica Acta 1793:636-649).

LAMP1 is highly expressed in late endosomes and lysosomes designatingLAMP1 as marker for these two organelles (Cook, N. R. et al., 2004,Traffic, 5 (9): 685-699). Thus, most of the literature on LAMP1 relatesto endocytosis, pinoscyosis, or phagocytosis (Cook, N. R. et al., 2004,Traffic, 5 (9): 685-699).

Although the majority of LAMP1 and LAMP2 reside in the lysosome, someLAMP1 and LAMP2 immunoreactivity is also observed at low levels at theplasma membrane. The LAMP1 found in the plasma membrane represents only1-2% of total LAMP1. This is for example true for peripheral bloodlymphocytes (Holcombe, R. F. et al. 1993, Clin Immunol Immunopathol.67(1): 31-39) and platelets (Silverstein, R. L. and Febbraio, M., 1992,Blood 80: 1470-1475).

Increased cell surface expression of LAMP1 and LAMP2 has been observedin tumor cell lines, for example in highly metastatic colonic carcinomaL4 cells (Saitoh, O. et al., 1992, J Biol Chem 267: 5700-5711), on humanmetastasizing melanoma A2058, HT1080 (human fibrosarcoma), CaCo-2 (humancolon-adenocarcinoma) cells and in colorectal neoplasms (Furuta, K. etal., 2001, J Pathol 159 (2): 449-455).

The chromosomal region 13q34 in which LAMP1 is located has recently beenlinked to amplification events including a larger amplicon that involvesCUL4A, LAMP1, TFDP1, and GAS6 in human breast cancer (Abba, Martin C. etal.; Cancer Res 2007; 4104). TFDP1 and perhaps CUL4A were identified inthe above mentioned publication as the leading genes driving theamplification phenomenon. In particular, analysis of publicly availablebreast cancer gene expression (microarrays) data sets indicated thatTFDP1 overexpression is associated with estrogen receptor (ER)-negativeand high-grade breast carcinomas, as well as shorter overall survival,relapse-free survival, and metastasis-free interval. Conversely, LAMP1expression did not significantly correlate with tumor grade. In the end,Abba et al. did not report that LAMP1 amplification translated intoLAMP1 overexpression in human breast cancer cells.

The 11 amino-acid cytoplasmic tail of LAMP1 contains a 7 amino-acidlinker sequence and a 4 amino acid long tyrosine motif (YQTI). It wasshown that small changes in the spacing of this motif relative to themembrane dramatically impair sorting in the early/sorting endosomes.Mutations within said tyrosine motif were shown to have an impact on thebinding of LAMP1 to adaptor proteins leading as well to impaired sorting(Obermüller, S. et al., 2002, Journal of Cell Science 115: 185-194;Rohrer, J. et al., 1996, Journal of Cell Biology 132(4): 565-576).Therefore, the abnormal cell surface expression of LAMP1 in differentcancer cell lines might be related to mutations in the cytoplasmic taileven though the mechanism is still unclear. Furthermore, it has beenshown that certain point mutations in the cytoplasmic tail lead toplasma membrane accumulation (Gough, N. R. et al., 1999, Journal of CellScience 112 (23): 4257-4269).

Due to the fact that LAMP1 is a marker for endosomes and lysosomes,numerous commercially available anti-LAMP1 antibodies were developed forresearch purposes. These antibodies are either polyclonal or monoclonaland are restricted to some biochemical application such asimmunohistochemistry (IHC), Western blots (WB), Fluorescence activatedcell sorter (FACS) analysis, Immunoprecipitation (IP) and Enzyme-linkedimmunosorbent assay (ELISA).

LAMP1 protein also has been detected at the cell membrane of tumorcells.

E. Venetsanakos (WO 2005/012912) suggested that LAMP1 is expressed onthe surface of colon cancer cells but not on the surface of normal coloncells and proposed that tumor growth might be reduced by targeting acytotoxic agent to LAMP1 via an anti-LAMP1 antibody. Venetsanakos didnot describe, however, preparation of anti-LAMP1 antibodies orconjugates thereof with cytotoxic or cytostatic agent or any datasupporting his hypothesis. Indeed, though a decade has passed sinceVenetsanakos' initial filing and no anti-LAMP1 antibodies or their useas immunoconjugates in an anti-LAMP1 therapy has entered clinicaldevelopment, so far. Accordingly, a great need exists for anti-LAMP1antibodies or immunoconjugates for the treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: sequence alignment of human and Macaca fascicularis LAMP1full-length proteins.

FIG. 2: Expression Profile of LAMP1 derived from FACS analysis with themonoclonal mouse antibodies Mab1 and MAb2.

FIG. 3: Reactivity of MAb1 with human LAMP1 and cynomolgus monkey LAMP1.

FIG. 4: Evaluation of the competition of MAb2 (murine) with MAb1(chimeric) for binding to LAMP1. With 2nd anti hFc being a secondaryantibody anti-human Fc and 2nd anti mFc being secondary antibodyanti-mouse Fc.

FIG. 5: Evaluation of the anti-tumor activity of DM4-SPDB-chMAb1conjugate against primary human colon adenocarcinoma CR-LRB-010P in SCIDfemale mice.

FIG. 6: Evaluation of the anti-tumor activity of DM4-SPDB-chMAb1conjugate against primary lung tumor LUN-NIC-0014 in SCID female mice.

FIG. 7: HRMS data of DM4-SPDB-chMAb1 conjugate.

FIG. 8A: Box Plot of RNA Intensity expression of LAMP1 by Copy NumberChanger category.

FIG. 8B: Plot of LAMP1 Copy Number according to LAMP1 mRNA expression oncolon tumors. Points represent individual mRNA expression, barscorresponds to mean values.

FIG. 9: A Sperman Correlation analysis of LAMP1 mRNA and Copy NumberChange data.

FIG. 10A: Box Plot of RNA Intensity expression and LAMP1 by Copy NumberChange in Soft Tissue Sarcoma.

FIG. 10B: Box Plot of RNA Intensity expression and LAMP1 by Copy NumberChange in Corpus Endometrioid Carcinoma.

FIG. 10C: Box Plot of RNA Intensity expression and LAMP1 by Copy NumberChange in Breast Invasive Carcinoma.

FIG. 11A: Histogram of LAMP1 Copy number by LAMP1 membrane expression(IHC category scoring) for colon tumor PDX.

FIG. 11B: Histogram of LAMP1 Copy number by LAMP1 membrane expression(IHC category scoring) for lung and stomach tumor PDXs.

FIG. 12: Expression profile oMAb1, 2 and 3 onto three PDXs (CR-IGR-034P,LUN-Nlc-014 and BRE-IGR-0159).

FIG. 13: Graphical representation showing the residues of Fab1 that arepart of the paratope (ie residues with atoms within 4A of the antigenatoms).

FIG. 14: Graphical representation showing the residues of hLAMP1 formingthe epitope for Fab1 (ie residues with atoms within 4A of the antigenatoms).

FIG. 15: Graphical representation showing the overlay of the residues ofhLAMP1 and a model of G187E corresponding to cLAMP1. Differences inorientation of Lys151 of hLAMP1 and of Tyr32 of Fab1-LC are indicated,necessary to accommodate the mutation from Glycine to Glutamine atposition 187.

FIG. 16: Graphical representation showing an overlay of the heavy chainresidues of Fab1 and a model of Fab1 with the mutation 1280 in its heavychain sequence of SEQ ID NO: 53 and the interaction with LAMP1.

FIG. 17: Graphical representation showing an overlay of the heavy chainresidues of Fab1 and a model of Fab1 with the mutation N55R in its heavychain sequence of SEQ ID NO: 53 and the interaction with LAMP1.

FIG. 18: HRMS data of DM4-SPDB-huMAb1_3 conjugate.

FIG. 19: HRMS data of DM4-SPDB-huMAb1_1 conjugate.

FIG. 20: HRMS data of DM4-SPDB-huMAb1_2 conjugate.

FIG. 21: HRMS data of DM4-SPDB-chMAb2 conjugate.

FIG. 22: HRMS data of DM4-SPDB-chMAb3 VLR24-R93 conjugate.

FIG. 23: Evaluation of the anti-tumor activity of DM4-SPDB-huMAb1_1against primary human colon adenocarcinoma CR-LRB-010P in SCID femalemice.

FIG. 24: Evaluation of the anti-tumor activity of DM4-SPDB-huMAb1_1against human primary invasive ductal carcinoma BRE-IGR-0159 in SCIDfemale mice.

FIG. 25A and FIG. 25B: Evaluation of the anti-tumor activity ofDM4-SPDB-huMAb1_1 against primary human lung tumor LUN-NIC-0070 in SCIDfemale mice.

FIG. 26: Evaluation of the anti-tumor activity of DM4-SPDB-chMAb2against primary human colon adenocarcinoma CR-LRB-010P in SCID femalemice.

FIG. 27: Evaluation of the anti-tumor activity of DM4-SPDB-chMAb2against human primary invasive ductal carcinoma BRE-IGR-0159 in SCIDfemale mice.

FIG. 28: Evaluation of the anti-tumor activity of DM4-SPDB-chMAb3against human primary invasive ductal carcinoma BRE-IGR-0159 in SCIDfemale mice.

FIG. 29: Graphical representation of the in vitro ADCC mediated bychMAb1, chMAb2 and chMAb3.

FIG. 30A through FIG. 30C: Graphical representation of the in vitro ADCCdependency on LAMP1 antigen density with HCT hu LAMP1 clone 8 LAMP1antigen denisty: 160000 (FIG. 30A), HCT hu LAMP1 clone 4 LAMP1 antigendenisty: 2000 (FIG. 30B), and HCT hu LAMP1 clone 12 LAMP1 antigendenisty: 5000 (FIG. 30C).

FIG. 31A: Comparison of in vitro ADCC of chMAb1 and DM4-SPDB-chMAb1.

FIG. 31B: Comparison of in vitro ADCC of chMAb2 and DM4-SPDB-chMAb2.

FIG. 32: In vitro ADCC mediated by huMAb1_1.

FIG. 33: In vitro ADCC mediated by DM4-SPDB-huMAb1_1.

FIG. 34A and FIG. 34B: Flow cytometry analysis of ADCP with a)Macrophages and target cells without Mab1_1 and b) Macrophages andtarget cells and Mab1_1.

FIG. 35: In vitro ADCP of huMAb1_negB.

FIG. 36: Loss of in vitro ADCP for huMAb1_negA.

FIG. 37: HRMS data of huMAb1_1 conjugate modified with SNPP with(2E,2′E,11aS,11a'S)-8,8′-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2,3-dihydro-1Hbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one)DM4-SPDB-chMAb2 conjugate.

FIG. 38: Imunohistochemistry staining (IHC) on FFPE sample of colonadenocarcinoma patient derived xenograft CR-LRB-010P and human breastcarcinoma with the polyclonal rabbit rAb4 Antibody. The Negativecontrols were performed by omission of the primary antibody.Furthermore, other irrelevant antibodies were negative or displayedintracellular immunostaining.

FIG. 39: Immunocytochemistry (ICC) in FFPE format with the polyclonalrabbit rAb4 Antibody at 1 μg/mL.

FIG. 40: Imunohistochemistry staining (IHC) on FFPE sample ofadenocarcinoma patient derived xenograft CR-LRB-010P with MAb4 obtainedfrom hybridoma 88LAMP1-2. The negative control was performed by omissionof the primary antibody.

FIG. 41: Binding affinity by ELISA of MAb4 towards LAMP1 (black) orLAMP2 (grey).

DETAILED DESCRIPTION Definitions

As used herein “LAMP1” designates the “Lysosomal associated membraneprotein 1”, a member of a family of glycoproteins that is also known asLAMPA, CD107a or LGP120. LAMP1 is, according to protein expression datafor human tumoral samples in comparison to non tumoral samples presentedin the following Example 5, expressed at the cell surface of colonadenocarcinomas, gastrointestinal tumors (small intestine, rectum,parotid gland), vital organs tumors (lung, liver, stomach, pancreas andkidney), reproductive organ tumors (breast, ovary and prostate) as wellas skin, larynx and soft tissue tumors.

The human gene LAMP1 is found on chromosome 13q34(113,951,469-113,977,441) and has a total length of 26,273 kb.

A reference sequence of the cDNA coding for full-length human LAMP1,including the sequence encoding the signal peptide, is available fromthe GenBank database under accession number NM_005561.3 (SEQ ID NO: 23)and the representative protein sequence, including the signal peptide(positions 1-28) is available under NP_005552.3 (SEQ ID NO: 24). Onepotential isoform of LAMP1 has been reported which would miss the aminoacids at positions 136-188 of SEQ ID NO: 24, corresponding to exon 4 ofthe gene coding for human LAMP1. No synonymous SNPs have been identifiedin Caucasian population of at least 60 individuals.

Concerning its orthologs, human LAMP1 shares 66% sequence identity withrespectively mouse LAMP1 (NP_034814, SEQ ID NO: 25) and rat LAMP1(NP_036989, SEQ ID NO: 26), and human and Macaca mulatta LAMP1(XP_001087801, SEQ ID NO: 27) share 96% sequence identity.

The sequence of LAMP1 from Macaca mulatta (SEQ ID NO: 27) and thepredicted sequence of Macaca fascicularis (SEQ ID NO: 39) are identicalto 99%, said sequences differing by one additional leucine at position11 of Macaca mulatta LAMP1 (SEQ ID NO: 27), i.e. in the signal peptide.Accordingly the sequences of mature LAMP1 from Macaca mulatta and Macacafascicularis are identical.

The closest member of the LAMP family is LAMP 2 (P13473, human LAMP2,soluble LAMP2 protein SEQ ID NO: 40). Human LAMP1 and LAMP2 proteinsshare ˜36% sequence identity, and comprise some conserved glycosylationsites.

A “domain” may be any region of a protein, generally defined on thebasis of sequence homologies and often related to a specific structuralor functional entity. The domain organization of LAMP1 has not beenentirely published so far.

Human LAMP1 consists of 417 amino acid residues and 28 amino-terminalresidues corresponding to a cleavable signal peptide. The major portionof LAMP1 resides on the lumenal side of the lysosome and is heavilyglycosylated by N-glycans. LAMP1 contains 18 potential N-glycosylationsites of which 5 are occupied with poly-N-acetyllactosamine glycans(Carlsson, S. R. and Fukuda, M., 1990, J. Biol. Chem. 265(33):20488-20495). They are clustered into two domains separated by ahinge-like structure enriched with prolines and serines many beinglinked to 0-glycans. LAMP1 has one transmembrane domain consisting of 24hydrophobic amino acids near the COOH terminus, and contains a shortcytoplasmic segment composed of 11 amino acid residues at theCOOH-terminal end.

The nomenclature of the two domains of LAMP1, “the first lumenal domain”and the “second lumenal domain” are based on the orientation of LAMP1within its original localization, the lysosome. Nevertheless, when LAMP1is expressed at the cell surface, the two lumenal domains becomeextracellular domains, and therefore exposed at the cell surface.Therefore, in one embodiment “extracellular” in context of the inventionrefers to LAMP1 protein constructs comprising the first and/or secondluminal domain(s) of LAMP1 as defined below and/or variants thereof. Thedomain organisation of human LAMP1 according to NP_005552.3 (SEQ ID NO:24) has been mapped in example 6.1 and will be used in this document asfollows:

TABLE 1 Description of human LAMP1 domains LAMP1 Domains Positions inNP_005552 (SEQ ID NO: 24) Peptide signal Met1-Ala28 First lumenal domainAla29-Arg195 Loop 1, L1 Ala29-Leu100 Loop 2, L2 Thr101-Arg195 HingePro196-Thr227 Second lumenal domain Asn228-Met382 Loop 3, L3Asn228-Ile309 Loop 4, L4 Leu310-Met382 Transmembrane domainLeu383-Gly406 Lysosome targeting motif Arg407-Ile417

Accordingly, the domain consisting of the first to third loops of humanLAMP1 consists of amino acids at positions 29-309 of SEQ ID NO: 24.

Domain organisation of Macaca fascicularis LAMP1 according to thepredicted sequence (SEQ ID NO: 39) is as follows:

TABLE 2 Description of Macaca fascicularis LAMP1 domains LAMP1 DomainsPositions in SEQ ID NO: 39 Peptide signal Met1-Ala26 First lumenaldomain Ala27-Arg193 Loop 1, L1 Ala27-L98 Loop 2, L2 Thr99-Arg193 HingePro194-Thr 225 Second lumenal domain Asn226-Met380 Loop 3, L3Asn226-Thr307 Loop 4, L4 Leu308-Met380 Transmembrane domainLeu381-Gly404 Lysosome targeting motif Arg405-Ile415

Accordingly, the domain consisting of first to third loops of Macacafascicularis LAMP1 consists of amino acids at positions 27-307 of SEQ IDNO: 39.

A sequence alignment of human and Macaca fascicularis LAMP1 full-lengthproteins is shown on FIG. 1.

The loop region 4 of human and Macaca fascicularis LAMP1 do not containany glycosylation site, which distinguishes Loop 4 from Loops 1-3 ofLAMP1.

Loops 1-4 have been defined from the primary amino acid sequence, andhas been mapped in example 6.1, but not from the 3D structure of LAMP1since the structure was not solved prior to this work.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon. A region encoding an expression product present in the DNAis called “coding DNA sequence” or “CDS”.

As used herein, references to specific proteins (e.g., antibodies) caninclude a polypeptide having a native amino acid sequence, as well asvariants and modified forms regardless of their origin or mode ofpreparation. A protein which has a native amino acid sequence is aprotein having the same amino acid sequence as obtained from nature.Such native sequence proteins can be isolated from nature or can beprepared using standard recombinant and/or synthetic methods. Nativesequence proteins specifically encompass naturally occurring truncatedor soluble forms, naturally occurring variant forms (e.g., alternativelyspliced forms), naturally occurring allelic variants and forms includingpost-translational modifications. A native sequence protein includesproteins following post-translational modifications such asglycosylation, or phosphorylation, or other modifications of some aminoacid residues.

As used herein, the term “marker” refers to any biological, chemical orphysical mean allowing identifying the presence, and possiblyquantifying the expression of a target gene and/or protein in abiological sample. Such markers are well known from one skilled in theart. Advantageously, the markers according to the invention are geneticmarkers and/or protein markers.

The term “gene” means a DNA sequence that codes for, or corresponds to,a particular sequence of amino acids which comprises all or part of oneor more proteins or enzymes, and may or may not include regulatory DNAsequences, such as promoter sequences, which determine for example theconditions under which the gene is expressed. Some genes, which are notstructural genes, may be transcribed from DNA to RNA, but are nottranslated into an amino acid sequence. Other genes may function asregulators of structural genes or as regulators of DNA transcription. Inparticular, the term gene may be intended for the genomic sequenceencoding a protein, i.e. a sequence comprising regulator, promoter,intron and exon sequences.

As used herein, the terms “copy number variation”, “copy number variant”and “CNV” are used indifferently and refer to a DNA segment of 1 kb orlarger and present at variable copy number in comparison with areference genome. The terms “structural variant”, “duplicon”, “indel”,“intermediate-sized structural variant (ISV)”, “low copy repeat (LCR)”,“multisite variant (MSV)”, “paralogous sequence variant (PSV)”,“segmental duplication”, “interchromosomal duplication”, and“intrachromosomal duplication”, found in the literature, are includedherein in the term “CNV”.

Furthermore, copy number variation can refer to a single gene, orinclude a contiguous set of genes.

As used herein “gene number” describes the numbers of genes present inthe cell. In diploid organisms, in a normal state, two copies of eachnucleic sequence are naturally present in the genome, therefore, thecopy number (CN) is =2. In particular, the genome displays two allelesfor each gene, one on each chromosome of a pair of homologouschromosomes (except for the genes localized on sexual chromosomes).

Herein the word “gene number” and “gene copy number” can be usedinterchangeably.

In the context of the invention, a “copy” of a sequence encompasses asequence identical to said sequence but also allelic variations of saidsequence.

One example to measure DNA copy number and therefore DNA copy numberchange is array-based CGH which is a high-throughput technique tomeasure DNA copy number change across the genome. The DNA fragments or“clones” of test and reference samples are hybridized to mapped arrayfragments. Log 2 intensity ratios of test to reference provide usefulinformation about genome-wide profiles in copy number.

The “Log 2” or “Log 2ratio” value is used to describe the copy number ofa gene or a DNA fragment in a cell genome. In an ideal situation, thelog 2 ratio of normal (copy-variation neutral) clones is log 2(2/2)=0,single copy losses is log 2(1/2)=−1, and single copy gains is log2(3/2)=0.58. Multiple copy gains or amplifications would have values oflog 2(4/2), log 2(5/2), . . . .

As used herein, the term “gain” of a sequence refers in general to thepresence of a copy number ≥2.5 (alternatively a Log 2ratio ≥0.32) ofsaid sequence in the diploid genome of a subject. These ≥2.5 copies maybe adjacent or not on the genome; in particular they may be present indifferent regions of a pair of chromosomes or on chromosomes belongingto distinct pairs of chromosomes of the genome.

Accordingly, the term “gene copy number gain” refers to the presence of≥2.5 copy numbers (alternatively a Log 2ratio ≥0.32) of a specific genein the diploid genome of a subject. When the copy number=2.5, 50% of thecells used for defining the copy number contain the usual 2 copies ofthe gene in a diploid organism and 50% of the cells used for definingthe copy number contain the usual 2 copies and 1 additional copy more ofsaid gene (in total 3 copies of said gene).

The term “low gain” of a sequence refers in general to the presence of acopy number ≥2.5 but <5 (alternatively 0.32≤ log 2 ratio <1.32) of saidsequence in the diploid genome of a subject. The terms “amplification”,“Amp”, or “high gain” refer herein to the presence of a copy number ≥5,or alternatively a Log 2≥1.32, of a specific sequence in the diploidgenome of a subject. Accordingly, the term “gene number amplification”refers to the presence of ≥5 copy numbers of a specific gene in thediploid genome of a subject

As used herein, a “fragment of a sequence” corresponds to a portion ofsaid sequence, for instance of a nucleotide sequence. Said fragment ispreferably at least 10 bp long. More preferably said fragment is atleast 15 bp long, in particular at least 20 bp long. Most preferably,said fragment is at least 25 bp long, at least 30 bp long, in particularat least 33 bp long. A fragment of the above sequence may be inparticular a primer or probe.

In the context of the invention, a “mutated sequence” of a referencesequence refers to a sequence including insertion(s), deletion(s) orsubstitution(s) of one or more nucleotide(s), wherein said mutatedsequence is at least 75% identical to the reference sequence. Thepercentage of sequence identity is calculated by comparing the mutatedsequence optimally aligned with the reference sequence, determining thenumber of positions at which the identical nucleic acid base (e.g., A,T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions of the reference sequence, and multiplying theresult by 100 to yield the percentage of sequence identity. Preferably,the mutated sequence is at least 80%, 85%, 90%, 95% identical to thereference sequence.

Preferably said mutated sequence of a reference sequence is an allelicvariant of said reference sequence. As used herein, an “allelic variant”denotes any of two or more alternative forms of a gene occupying thesame chromosome locus.

A sequence “at least 85% identical to a reference sequence” is asequence having, on its entire length, 85%, or more, in particular 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with theentire length of the reference sequence.

A percentage of “sequence identity” may be determined by comparing thetwo sequences, optimally aligned over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e. gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for comparison is conducted byglobal pairwise alignment, e.g. using the algorithm of Needleman andWunsch J. Mol. Biol. 48: 443 (1970). The percentage of sequence identitycan be readily determined for instance using the program Needle, withthe BLOSUM62 matrix, and the following parameters gap-open=10,gap-extend=0.5.

A “conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chainR group with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartic acid and glutamic acid; and 7)sulfur-containing side chains: cysteine and methionine. Conservativeamino acids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine-tryptophane, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine.

An “antibody” may be a natural or conventional antibody in which twoheavy chains are linked to each other by disulfide bonds and each heavychain is linked to a light chain by a disulfide bond. There are twotypes of light chain, lambda (I) and kappa (κ). There are five mainheavy chain classes (or isotypes) which determine the functionalactivity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chaincontains distinct sequence domains. The light chain includes two domainsor regions, a variable domain (VL) and a constant domain (CL). The heavychain includes four domains, a variable domain (VH) and three constantdomains (CH1, CH2 and CH3, collectively referred to as CH). The variableregions of both light (VL) and heavy (VH) chains determine bindingrecognition and specificity to the antigen. The constant region domainsof the light (CL) and heavy (CH) chains confer important biologicalproperties such as antibody chain association, secretion,trans-placental mobility, complement binding, and binding to Fcreceptors (FcR). The Fv fragment is the N-terminal part of the Fabfragment of an immunoglobulin and consists of the variable portions ofone light chain and one heavy chain. The specificity of the antibodyresides in the structural complementarity between the antibody combiningsite and the antigenic determinant. Antibody combining sites are made upof residues that are primarily from the hypervariable or complementaritydetermining regions (CDRs). Occasionally, residues from nonhypervariableor framework regions (FR) influence the overall domain structure andhence the combining site. Complementarity Determining Regions or CDRsrefer to amino acid sequences which together define the binding affinityand specificity of the natural Fv region of a native immunoglobulinbinding site. The light and heavy chains of an immunoglobulin each havethree CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H,CDR3-H, respectively. A conventional antibody antigen-binding site,therefore, includes six CDRs, comprising the CDR set from each of aheavy and a light chain V region.

“Framework Regions” (FRs) refer to amino acid sequences interposedbetween CDRs, i.e. to those portions of immunoglobulin light and heavychain variable regions that are relatively conserved among differentimmunoglobulins in a single species. The light and heavy chains of animmunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L,FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.

As used herein, a “human framework region” is a framework region that issubstantially identical (about 85%, or more, in particular 90%, 95%,97%, 99% or 100%) to the framework region of a naturally occurring humanantibody.

-   -   In the context of the invention, CDR/FR definition in an        immunoglobulin light or heavy chain is to be determined based on        IMGT definition (Lefranc, M. P. et al., 2003, Dev Comp Immunol.        27(1): 55-77; www.imgt.org).

As used herein, the term “antibody” denotes conventional antibodies andfragments thereof, as well as single domain antibodies and fragmentsthereof, in particular variable heavy chain of single domain antibodies,and chimeric, humanised, bispecific or multispecific antibodies.

As used herein, antibody or immunoglobulin also includes “single domainantibodies” which have been more recently described and which areantibodies whose complementary determining regions are part of a singledomain polypeptide. Examples of single domain antibodies include heavychain antibodies, antibodies naturally devoid of light chains, singledomain antibodies derived from conventional four-chain antibodies,engineered single domain antibodies. Single domain antibodies may bederived from any species including, but not limited to mouse, human,camel, llama, goat, rabbit and bovine. Single domain antibodies may benaturally occurring single domain antibodies known as heavy chainantibody devoid of light chains. In particular, Camelidae species, forexample camel, dromedary, llama, alpaca and guanaco, produce heavy chainantibodies naturally devoid of light chain. Camelid heavy chainantibodies also lack the CH1 domain.

The variable heavy chain of these single domain antibodies devoid oflight chains are known in the art as “VHH” or “nanobody”. Similar toconventional VH domains, VHHs contain four FRs and three CDRs.Nanobodies have advantages over conventional antibodies: they are aboutten times smaller than IgG molecules, and as a consequence properlyfolded functional nanobodies can be produced by in vitro expressionwhile achieving high yield. Furthermore, nanobodies are very stable, andresistant to the action of proteases. The properties and production ofnanobodies have been reviewed by Harmsen and De Haard H J (Appl.Microbiol. Biotechnol. 2007 November; 77(1): 13-22).

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody molecule of a single amino acid composition that is directedagainst a specific antigen, and is not to be construed as requiringproduction of the antibody by any particular method. A monoclonalantibody may be produced by a single clone of B cells or hybridoma, butmay also be recombinant, i.e. produced by protein engineering.

The term “chimeric antibody” refers to an engineered antibody which inits broadest sense contains one or more regions from one antibody andone or more regions from on or more other antibody(ies). In particular achimeric antibody comprises a VH domain and a VL domain of an antibodyderived from a non-human animal, in association with a CH domain and aCL domain of another antibody, in particular a human antibody. As thenon-human animal, any animal such as mouse, rat, hamster, rabbit or thelike can be used. A chimeric antibody may also denote a multispecificantibody having specificity for at least two different antigens. In anembodiment, a chimeric antibody has variable domains of mouse origin andconstant domains of human origin

The term “humanised antibody” refers to an antibody which is initiallywholly or partially of non-human origin and which has been modified toreplace certain amino acids, in particular in the framework regions ofthe heavy and light chains, in order to avoid or minimize an immuneresponse in humans. The constant domains of a humanized antibody aremost of the time human CH and CL domains. In an embodiment, a humanizedantibody has constant domains of human origin.

“Fragments” of (conventional) antibodies comprise a portion of an intactantibody, in particular the antigen binding region or variable region ofthe intact antibody. Examples of antibody fragments include Fv, Fab,F(ab′)2, Fab′, dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific andmultispecific antibodies formed from antibody fragments. A fragment of aconventional antibody may also be a single domain antibody, such as aheavy chain antibody or VHH.

The term “Fab” denotes an antibody fragment having a molecular weight ofabout 50,000 and antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating IgG with a protease, papaine, are bound togetherthrough a disulfide bond.

The term “F(ab′)2” refers to an antibody fragment having a molecularweight of about 100,000 and antigen binding activity, which is slightlylarger than the Fab bound via a disulfide bond of the hinge region,among fragments obtained by treating IgG with a protease, pepsin.

A single chain Fv (“scFv”) polypeptide is a covalently linked VH::VLheterodimer which is usually expressed from a gene fusion including VHand VL encoding genes linked by a peptide-encoding linker. The humanscFv fragment of the invention includes CDRs that are held inappropriate conformation, in particular by using gene recombinationtechniques. Divalent and multivalent antibody fragments can form eitherspontaneously by association of monovalent scFvs, or can be generated bycoupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)₂.“dsFv” is a VH::VL heterodimer stabilised by a disulphide bond.“(dsFv)2” denotes two dsFv coupled by a peptide linker.

The term “bispecific antibody” or “BsAb” denotes an antibody whichcombines the antigen-binding sites of two antibodies within a singlemolecule. Thus, BsAbs are able to bind two different antigenssimultaneously. Genetic engineering has been used with increasingfrequency to design, modify, and produce antibodies or antibodyderivatives with a desired set of binding properties and effectorfunctions as described for instance in EP 2 050 764 A1.

The term “multispecific antibody” denotes an antibody which combines theantigen-binding sites of two or more antibodies within a singlemolecule.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

The term “hybridoma” denotes a cell, which is obtained by subjecting a Bcell prepared by immunizing a non-human mammal with an antigen to cellfusion with a myeloma cell derived from a mouse or the like whichproduces a desired monoclonal antibody having an antigen specificity.

By “purified” and “isolated” it is meant, when referring to apolypeptide (i.e. the antibody of the invention) or a nucleotidesequence, that the indicated molecule is present in the substantialabsence of other biological macromolecules of the same type. The term“purified” as used herein in particular means at least 75%, 85%, 95%, or98% by weight, of biological macromolecules of the same type arepresent. An “isolated” nucleic acid molecule which encodes a particularpolypeptide refers to a nucleic acid molecule which is substantiallyfree of other nucleic acid molecules that do not encode the subjectpolypeptide; however, the molecule may include some additional bases ormoieties which do not deleteriously affect the basic characteristics ofthe composition.

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. In particular a subject according tothe invention is a human.

Throughout the instant application, the term “comprising” is to beinterpreted as encompassing all specifically mentioned features as welloptional, additional, unspecified ones. As used herein, the use of theterm “comprising” also discloses the embodiment wherein no featuresother than the specifically mentioned features are present (i.e.“consisting of”).

Throughout the instant application, the term “and/or” is a grammaticalconjunction that is to be interpreted as encompassing that one or moreof the cases it connects may occur. For example, the sentence“quantifying the expression of a target gene and/or protein in abiological sample” indicates the expression of a target gene may bequantified (mRNA), or the expression of a protein or the expression of atarget gene (mRNA) and the protein together may be quantified.

Accordingly, the wording “a variable domain of heavy chain of sequenceSEQ ID NO: 1 or a sequence at least 85% identical thereto and/or avariable domain of light chain of sequence of sequence SEQ ID NO: 5, ora sequence at least 85% identical thereto” is to b interpreted as “avariable domain of heavy chain of sequence SEQ ID NO: 1 or a sequence atleast 85% identical thereto” or “a variable domain of light chain ofsequence of sequence SEQ ID NO: 5, or a sequence at least 85% identicalthereto” or “a variable domain of heavy chain of sequence SEQ ID NO: 1or a sequence at least 85% identical thereto and a variable domain oflight chain of sequence of sequence SEQ ID NO: 5, or a sequence at least85% identical thereto”.

The term “cancer”, “neoplasm”, “tumor”, and “carcinoma” are usedinterchangeably herein to refer to cells that exhibit relativelyautonomous growth, so that they can exhibit an aberrant growth phenotypecharacterized by significant loss of control of cell proliferation. Ingeneral, cells of interest for detection or treatment in the presentapplication include precancerous (e.g. benign), malignant, metastatic,and non-metastatic cells.

Immunoconjugates

For therapeutic purposes, it is advantageous to create an antibody withoptimal characteristics for use as an antibody drug conjugate, i.e. anantibody which specifically recognizes a target present on the surfaceof cancer cells and which is capable of efficiently triggeringinternalization once bound to said target.

The inventors raised antibodies against colon tumor cells or lung tumorcells and screened resulting clones for the differential binding totumor cells and non-tumor tissue.

The inventors identified in this way antibodies distinguishing tumoralfrom non-tumoral tissues. Three of those antibodies were selected (theso-called antibodies “MAb1”, “MAb2” and “MAb3”), fulfilling the expectedfeatures necessary for therapeutical application, in particular in theform of ADC. Those three antibodies showed high binding affinity (withinthe nanomolar range) to cell surface expressed LAMP1 in cancer cells.Furthermore, those three anti-LAMP1 antibodies showed high capacity totrigger internalization of the LAMP1/anti-LAMP1 antibody complex, asshown in example 4.4 and 4.3.

The inventors demonstrated that the chimeric antibodies derived fromMAb1, MAb2, MAb3 (chMAb1, chMAb2, chMAb3), combined with a cytotoxicmaytansinoid (DM4) showed as well a high but slightly different bindingaffinity to human LAMP1 or cynomologus monkey LAMP1 then the nakedantibody as shown in example 8.1.7 and 8.1.8.

Accordingly in one embodiment the immunoconjugate in context of theinvention has an affinity (EC₅₀) for full length human LAMP1 andcynomologues monkey LAMP1 expressed at the cell surface of a recombinantcell line, wherein the cell line may be HCT116 and the apparent affinitymeasured via Flow Cytometry is ≤30 nM, for example ≤20 nM or ≤15 nM.

The Methods to measure the affinity (EC₅₀) for full length human LAMP1and cynomologues monkey LAMP1 are further explained in the chapter“antibodies”.

The inventors additionally demonstrated that a chimeric antibody derivedfrom MAb1 (chMAb1), combined with a cytotoxic maytansinoid (DM4),induces cytotoxic activity in vitro on human HCT116 tumor cellscontaining a stable integration of the LAMP1 coding DNA sequence in thegenomic DNA and expressing LAMP1 on their surface.

Furthermore, the inventors demonstrated that humanized antibodiesderived from MAb1 (huMAb1_1, huMAb1_2, huMAb1_3), combined with acytotoxic maytansinoid (DM4) induce cytotoxic activity in vitro on humanHCT116 tumor cells containing a stable integration of the LAMP1 codingDNA sequence in the genomic DNA.

They have also shown that the immunoconjugate DM4-SPDB-chMAb1 induces amarked anti-tumor activity in vivo in mice bearing the primary humancolon adenocarcinoma xenograft derived from patient CR-LRB-010P, whenused at a dose of 10 mg/kg, 5 mg/kg and 2.5 mg/kg, with a singleinjection, as described in example 10.1.1.

Furthermore, the inventors showed that this immunoconjugate induces amarked anti-tumor activity in vivo in mice bearing the primary humanlung tumor xenograft derived from patient LUN-NIC-0014, when used at adose of 10 mg/kg, 5 mg/kg and 2.5 mg/kg, with a single injection, asdescribed in example 10.1.2.

They have also shown that the immunoconjugates DM4-SPDB-huMAb1_1,DM4-SPDB-chMAb2, and DM4-SPDB-chMAb3 induce a marked anti-tumor activityin vivo in different patient-derived xenograft as shown in example10.2-10.4.

For example, it was shown the immunoconjugate DM4-SPDB-huMAb1_1 inducesa marked anti-tumor activity in vivo in a primary human invasive ductalcarcinoma xenograft and primary human lung tumor xenograft derived frompatient, when used at a dose of 10 mg/kg, 5 mg/kg, 2.5 mg/kg, or 1.25mg/kg with a single injection, as described in example 10.2.2 and10.2.3.

Also the immunoconjugates DM4-SPDB-chMAb2 and DM4-SPDB-chMAb3 induced amarked anti-tumor activity in vivo in a murine model of primary humaninvasive ductal carcinoma xenograft derived from patient, when used at adose of 10 mg/kg, 5 mg/kg and 2.5 mg/kg or 5 mg/kg, 2.5 mg/kg and 1.25mg/kg, respectively, with a single injection, as described in example10.3.2 and 10.4.

Altogether, for the first time, these results validly identify LAMP1 asa therapeutic target for the treatment of cancer.

Accordingly, the invention relates to an immunoconjugate comprising anantibody which:

-   -   a) binds to human and Macaca fascicularis LAMP1 proteins; and    -   b) is linked or conjugated to at least one growth inhibitory        agent.

Any antibody which binds to human and Macaca fascicularis LAMP1proteins, as described throughout the instant application (e.g. MAb4,fragments thereof, or chimeric or humanised version thereof), can beincorporated in the immunoconjugate according to the invention.

As used herein, “conjugate”, “immunoconjugate”, “antibody-drugconjugate” or “ADC” have the same meaning and are interchangeable.

A “growth inhibitory agent”, or “anti-proliferative agent”, which can beused indifferently, refers to a compound or composition which inhibitsgrowth of a cell, especially tumour cell, either in vitro or in vivo. Agrowth inhibitory agent denotes in particular a cytotoxic agent or aradioactive isotope.

The term “radioactive isotope” is intended to include radioactiveisotopes suitable for treating cancer, such as At²¹¹, Ac²²⁵, Bi²¹²,Bi²¹³, Pb²¹², Er¹⁶⁹, I¹³¹, I¹²⁴, I¹²⁵, Y⁹⁰, In¹¹¹, P³², Re¹⁸⁶, Re¹⁸⁸,Sm¹⁵³, Sr⁸⁹, Zr⁸⁹, Tc^(99m), Ga⁶⁸, Cu⁸⁴ and radioactive isotopes of Lusuch as Lu¹⁷⁷. Such radioisotopes generally emit mainly beta-radiation.In an embodiment the radioactive isotope is alpha-emitter isotope, moreprecisely Thorium 227 (Th²²⁷) which emits alpha-radiation. Theimmunoconjugates according to the present invention can be prepared asdescribed in the application WO2004/091668.

In one embodiment, a radioactive isotope is selected from the groupconsisting of At²¹¹, Ac²⁵, Bi²¹³, Pb²¹², Er¹⁶⁹, I¹²⁴, I¹²⁵, In¹¹¹, P³²,Re¹⁸⁶, Sm¹⁵³, Sr⁸⁹, Zr⁸⁹, Tc^(99m), Ga⁶⁸, Cu⁶⁴ and radioactive isotopesof Lu, for instance from At²¹¹, Er¹⁶⁹, I¹²⁵, In¹¹¹, P³², Re¹⁸⁶, Sm¹⁵³,Sr⁸⁹, radioactive isotopes of Lu, and Th²²⁷.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term “cytotoxic agent” is intended to includechemotherapeutic agents, enzymes, antibiotics, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof, andthe various antitumor or anticancer agents disclosed below. In someembodiments, the cytotoxic agent is a drug or a pro-drug of a compoundconsisting in an anti-tubulin agent such as taxoids or taxanes, avinca-alkaloid, a maytansinoid or maytansinoid analog such as DM1 orDM4, a cryptophycin derivative, an auristatin or dolastatin analog; aDNA alkylating agent, such as a tomaymycin or pyrrolobenzodiazepinederivative, a CC-1065 or CC-1065 analog; a leptomycin derivative; atopoisomerase II inhibitor, an RNA polymerase II inhibitor such asalpha-amanitin.

According to a first embodiment, said at least one growth inhibitoryagent is neither an undefined radioactive isotope, a chemotherapeuticdrug, a protein or lectin, nor pokeweed antiviral protein, abrin, ricinand each of their A chains, doxorubicin, cisplastin, Iodine-131,Yttrium-90, Rhenium-188, Bismuth-212, Taxol, 5-Fluorouracil, VP-16(etoposide), bleomycin, methotrexate, vindesine, adriamycin,vincristine, vinblastine, bis-chloroethylnitrosourea (BCNU), mitomycin,cyclophosphamide and a cytokine such as TNF and TNF-β.

According to this first embodiment, the invention relates in particularto an immunoconjugate comprising an antibody which:

-   a) binds to human and Macaca fascicularis LAMP1 proteins; and-   b) is linked or conjugated to at least one growth inhibitory agents    -   (i) a cytotoxic agent selected from the group consisting of        enzymes other than from pokeweed antiviral protein; antibiotics        other than from bleomycin and mitomycin; toxins of bacterial,        fungal, or animal origin or of plant origin other than from        abrin and ricin, including fragments and/or variants thereof; a        drug or a pro-drug of a compound consisting in an anti-tubulin        agent such as a maytansinoid or maytansinoid analog such as DM1        or DM4, a taxoid or taxane other than from paclitaxel (Taxol), a        vinca-alkaloid other than from vindesine, vincristine and        vinblastine, a cryptophycin derivative, an auristatin or        dolastatin analog; a DNA alkylating agent other than from BCNU        and cyclophosphamide, such as a tomaymycin or        pyrrolobenzodiazepine derivative, a CC-1065 or CC-1065 analog; a        leptomycin derivative; a topoisomerase II inhibitors other than        doxorubicin (adriamycin) and etoposide, a RNA polymerase II        inhibitor such as alpha-amanitin, or    -   (ii) a radioactive isotope selected from the group consisting of        At²¹¹, Ac²²⁵, Bi²¹³, Pb²¹², Er¹⁶⁹, I¹²⁴, I¹²⁵, In¹¹¹, P³²,        Re¹⁸⁶, Sm¹⁵³, Sr⁸⁹, Zr⁸⁹, Tc^(99m), Ga⁶⁸, Cu⁶⁴ and radioactive        isotopes of Lu such as Lu¹⁷⁷, and Th²²⁷.

In one embodiment a radioactive isotope is selected from the groupconsisting of At²¹¹, Er¹⁶⁹, I¹²⁵, In¹¹¹, P³², Re¹⁸⁶, Sm¹⁵³, Sr⁸⁹,radioactive isotopes of Lu, and Th²²⁷.

In said first embodiment, the antibody may bind in particular to adomain consisting of the first to third loops of human and Macacafascicularis LAMP1 proteins; wherein the domain consisting of the firstto third loops of human LAMP1 protein consists of amino acids Ala29 toIle309 of SEQ ID NO: 24 and the domain consisting of the first to thirdloops of Macaca fascicularis LAMP1 protein consists of amino acids Ala27to Thr307 of SEQ ID NO: 39

According to a second embodiment, the invention relates to animmunoconjugate wherein the antibody binds to a domain consisting of thefirst to third loops of human and Macaca fascicularis LAMP1 proteins;wherein the domain consisting of the first to third loops of human LAMP1protein consists of amino acids Ala29 to Ile309 of SEQ ID NO: 24 and thedomain consisting of the first to third loops of Macaca fascicularisLAMP1 protein consists of amino acids Ala27 to Thr307 of SEQ ID NO: 39.

Therefore, according to this second embodiment, the immunoconjugatecomprises an antibody which

-   a) binds to a domain consisting of the first to third loops of human    and Macaca fascicularis LAMP1 proteins; wherein the domain    consisting of the first to third loops of human LAMP1 protein    consists of amino acids Ala29 to Ile309 of SEQ ID NO: 24 and the    domain consisting of the first to third loops of Macaca fascicularis    LAMP1 protein consists of amino acids Ala27 to Thr307 of SEQ ID NO:    39; and-   b) is linked or conjugated to said at least one growth inhibitory    agent.

Although not compulsory, in said second embodiment, the at least onegrowth inhibitory agent may be different from an undefined radioactiveisotope, a chemotherapeutic drug, a protein or lectin, in particularfrom pokeweed antiviral protein, abrin, ricin and each of their Achains, doxorubicin, cisplastin, Iodine-131, Yttrium-90, Rhenium-188,Bismuth-212, Taxol, 5-Fluorouracil, VP-16 (etoiposide), bleomycin,methotrexate, vindesine, adriamycin, vincristine, vinblastine, BCNU,mitomycin, cyclophosphamide and a cytokine such as TNF and TNF-β.

Accordingly, said at least one growth inhibitory agent may be aradioactive isotopes selected from the group consisting of At²¹¹, Ac²²⁵,Bi²¹³, Pb²¹², Er¹⁶⁹, I¹²⁴, I¹²⁵, In¹¹¹, P³², Re¹⁸⁶, Sm¹⁵³, Sr⁸⁹, Zr⁸⁹,Tc^(99m), Ga⁶⁸, Cu⁶⁴ and radioactive isotopes of Lu such as Lu¹⁷⁷, andTh²²⁷, for instance At²¹¹, Er¹⁶⁹, I¹²⁵, In¹¹¹, P³², Re¹⁸⁶, Sm¹⁵³, Sr⁸⁹,radioactive isotopes of Lu such as Lu¹⁷⁷, and Th²²⁷, or a cytotoxicagent as defined in said first embodiment.

In said first and second embodiments, said at least one growthinhibitory agent may be in particular drug or a pro-drug of a compoundconsisting in a maytansinoid or maytansinoid analog such as DM1 or DM4,a tomaymycin or pyrrolobenzodiazepine derivative, a cryptophycinderivative, a leptomycin derivative, an auristatin or dolastatin analog,or a CC-1065 or CC-1065 analog, a RNA polymerase II inhibitor such asalpha-amanitin.

In one embodiment, a suitable tomamycin is a tomamycine dimer. Saidtomamycin dimer is for instance(2E,2′E,11aS,11a'S)-8,8′-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2,3-dihydro-1Hbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one).

The structural formula of(2E,2′E,11aS,11a'S)-8,8′-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2,3-dihydro-1Hbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one) is

A “maytansinoid” as used herein denotes maytansinoids and maytansinoidanalogs. Maytansinoids are drugs that inhibit microtubule formation andthat are highly toxic to mammalian cells.

Examples of suitable maytansinoids include maytansinol and maytansinolanalogs.

Examples of suitable maytansinol analogues include those having amodified aromatic ring and those having modifications at otherpositions. Such suitable maytansinoids are disclosed in U.S. Pat. Nos.4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Specific examples of suitable analogues of maytansinol having a modifiedaromatic ring include:

-   -   (1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH        reduction of ansamytocin P2);    -   (2) C-20-hydroxy (or C-20-demethyl) +/−C-19-dechloro (U.S. Pat.        Nos. 4,361,650 and 4,307,016) (prepared by demethylation using        Streptomyces or Actinomyces or dechlorination using LAH); and    -   (3) C-20-demethoxy, C-20-acyloxy (−OCOR), +/−dechloro (U.S. Pat.        No. 4,294,757) (prepared by acylation using acyl chlorides).

Specific examples of suitable analogues of maytansinol havingmodifications of other positions include:

-   -   (1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction        of maytansinol with H₂S or P₂S₅);    -   (2) C-14-alkoxymethyl (demethoxy/CH₂OR) (U.S. Pat. No.        4,331,598);    -   (3) C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S.        Pat. No. 4,450,254) (prepared from Nocardia);    -   (4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by        the conversion of maytansinol by Streptomyces);    -   (5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929)        (isolated from Trewia nudiflora);    -   (6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348)        (prepared by the demethylation of maytansinol by Streptomyces);        and    -   (7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the        titanium trichloride/LAH reduction of maytansinol).

In a specific embodiment, the cytotoxic conjugates of the presentinvention utilize the thiol-containing maytansinoid (DM1), formallytermed N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine, asthe cytotoxic agent. DM1 is represented by the following structuralformula (I):

In another embodiment, the cytotoxic conjugates of the present inventionutilize the thiol-containing maytansinoid DM4, formally termedN^(2′)-deacetyl-N^(2′)-(4-methyl-4-mercapto-1-oxopentyl)-maytansine, asthe cytotoxic agent. DM4 is represented by the following structuralformula (II):

In further embodiments of the invention, other maytansines, includingthiol and disulfide-containing maytansinoids bearing a mono or di-alkylsubstitution on the carbon atom bearing the sulfur atom, may be used.These include a maytansinoid having, at C-3, C-14 hydroxymethyl, C-15hydroxy, or C-20 desmethyl, an acylated amino acid side chain with anacyl group bearing a hindered sulfhydryl group, wherein the carbon atomof the acyl group bearing the thiol functionality has one or twosubstituents, said substituents being CH₃, C₂H₅, linear or branchedalkyl or alkenyl having from 1 to 10 reagents and any aggregate whichmay be present in the solution.

Examples of these cytotoxic agents and of methods of conjugation arefurther given in the application WO2008/010101 which is incorporated byreference.

In some embodiments of the present invention, the antibody is covalentlyattached, directly or via a cleavable or non-cleavable linker, to the atleast one growth inhibitory agent.

“Linker”, as used herein, means a chemical moiety comprising a covalentbond or a chain of atoms that covalently attaches a polypeptide to adrug moiety.

The conjugates may be prepared by in vitro methods. In order to link adrug or prodrug to the antibody, a linking group is used. Suitablelinking groups are well known in the art and include disulfide groups,thioether groups, acid labile groups, photolabile groups, peptidaselabile groups and esterase labile groups. Conjugation of an antibody ofthe invention with cytotoxic agents or growth inhibitory agents may bemade using a variety of bifunctional protein coupling agents includingbut not limited to N-succinimidyl pyridyldithiobutyrate (SPDB), butanoicacid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester(nitro-SPDB), 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid(sulfo-SPDB), N-succinimidyl (2-pyridyldithio) propionate (SPDP), SNPP(N-succinimidyl 4-(5-nitro-2-pyridyldithio)pentanoate), succinimidyl(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al (1987). Carbon labeled1-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody (WO 94/11026).

The linker may be a “cleavable linker” facilitating release of thecytotoxic agent or growth inhibitory agent in the cell. For example, anacid-labile linker, a peptidase-sensitive linker, an esterase labilelinker, a photolabile linker or a disulfide-containing linker (See e.g.U.S. Pat. No. 5,208,020) may be used. The linker may be also a“non-cleavable linker” (for example SMCC linker) that might lead tobetter tolerance in some cases.

Alternatively, a fusion protein comprising the antibody of the inventionand a cytotoxic or growth inhibitory polypeptide may be made, byrecombinant techniques or peptide synthesis. The length of DNA maycomprise respective regions encoding the two portions of the conjugateeither adjacent one another or separated by a region encoding a linkerpeptide which does not destroy the desired properties of the conjugate.

The antibodies of the present invention may also be used in DependentEnzyme Mediated Prodrug Therapy by conjugating the polypeptide to aprodrug-activating enzyme which converts a prodrug (e.g. a peptidylchemotherapeutic agent, see WO81/01145) to an active anti-cancer drug(See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278). The enzymecomponent of the immunoconjugate useful for ADEPT includes any enzymecapable of acting on a prodrug in such a way so as to convert it intoits more active, cytotoxic form. Enzymes that are useful in the methodof this invention include, but are not limited to, alkaline phosphataseuseful for converting phosphate-containing prodrugs into free drugs;arylsulfatase useful for converting sulfate-containing prodrugs intofree drugs; cytosine deaminase useful for converting non-toxicfluorocytosine into the anticancer drug, 5-fluorouracil; proteases, suchas serratia protease, thermolysin, subtilisin, carboxypeptidases andcathepsins (such as cathepsins B and L), that are useful for convertingpeptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases,useful for converting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as O-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; P-lactamaseuseful for converting drugs derivatized with P-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. The enzymes can be covalently bound to the polypeptides ofthe invention by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above.

According to said first and second embodiments, in the conjugate of theinvention, the growth inhibitory agent may be a maytansinoid, inparticular DM1 or DM4.

In such a conjugate, the antibody is conjugated to said at least onegrowth inhibitory agent by a linking group. In particular said linkinggroup is a non-cleavable linker, such as SPDB, sulfo-SPDB, or SMCC.

In particular, the conjugate may be selected from the group consistingof:

-   -   i) an antibody-SPDB-DM4 conjugate of formula (III)

-   -   ii) an antibody-sulfo-SPDB-DM4 conjugate of formula (IV)

-   -   iii) an antibody-SMCC-DM1 conjugate of formula (V)

In a further embodiment, in the conjugate of the invention, the growthinhibitory agent may be tomamycin, for instance a tomamycin dimer, forexample(2E,2′E,11aS,11a'S)-8,8′-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2,3-dihydro-1Hbenzo[e]pyrrolo[1,2-a][1,4] diazepin-5(11aH)-one).

In such a conjugate, the antibody is conjugated to said at least onegrowth inhibitory agent by a linking group, for instance by SNPP.

Accordingly, in one embodiment the conjugate may anantibody-SNPP-(2E,2′E,11aS,11a'S)-8,8′-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl).

In general, the conjugate can be obtained by a process comprising thesteps of:

-   -   (i) bringing into contact an optionally-buffered aqueous        solution of a cell-binding agent (e.g. an antibody according to        the invention) with solutions of a linker and a cytotoxic        compound;    -   (ii) then optionally separating the conjugate which was formed        in (i) from the unreacted cell-binding agent.

The aqueous solution of cell-binding agent can be buffered with bufferssuch as, e.g. potassium phosphate, acetate, citrate orN-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes buffer). Thebuffer depends upon the nature of the cell-binding agent. The cytotoxiccompound is in solution in an organic polar solvent, e.g. dimethylsulfoxide (DMSO) or dimethylacetamide (DMA).

The reaction temperature is usually comprised between 20° C. and 40° C.The reaction time can vary from 1 to 24 hours. The reaction between thecell-binding agent and the cytotoxic agent can be monitored by sizeexclusion chromatography (SEC) with a refractometric and/or UV detector.If the conjugate yield is too low, the reaction time can be extended.

A number of different chromatography methods can be used by the personskilled in the art in order to perform the separation of step (ii): theconjugate can be purified e.g. by SEC, adsorption chromatography (suchas ion exchange chromatography, IEC), hydrophobic interactionchromatograhy (HIC), affinity chromatography, mixed-supportchromatography such as hydroxyapatite chromatography, or highperformance liquid chromatography (HPLC). Purification by dialysis ordiafiltration can also be used.

As used herein, the term “aggregates” means the associations which canbe formed between two or more cell-binding agents, said agents beingmodified or not by conjugation. The aggregates can be formed under theinfluence of a great number of parameters, such as a high concentrationof cell-binding agent in the solution, the pH of the solution, highshearing forces, the number of bonded dimers and their hydrophobiccharacter, the temperature (see Wang, L. and Gosh, R., 2008, J. MembrSci. 318: 311-316, and references cited therein); note that the relativeinfluence of some of these parameters is not clearly established. In thecase of proteins and antibodies, the person skilled in the art willrefer to Cromwell, M. E. et al. (2006, AAPS Jounal 8(3): E572-E579). Thecontent in aggregates can be determined with techniques well known tothe skilled person, such as SEC (see Walter et al., 1993, Anal.Biochem., 212(2): 469-480).

After step (i) or (ii), the conjugate-containing solution can besubmitted to an additional step (iii) of chromatography, ultrafiltrationand/or diafiltration.

The conjugate is recovered at the end of these steps in an aqueoussolution.

According to an embodiment, the conjugate according to the invention ischaracterised by a “drug-to-antibody ratio” (or “DAR”) as measured byDAR UV ranging from 1 to 10, for instance from 2 to 5, in particularfrom 3 to 4. This is generally the case of conjugates includingmaytansinoid molecules.

This DAR number can vary with the nature of the antibody and of the drug(i.e. the growth-inhibitory agent) used along with the experimentalconditions used for the conjugation (like the ratio growth-inhibitoryagent/antibody, the reaction time, the nature of the solvent and of thecosolvent if any). Thus the contact between the antibody and thegrowth-inhibitory agent leads to a mixture comprising several conjugatesdiffering from one another by different drug-to-antibody ratios;optionally the naked antibody; optionally aggregates. The DAR that isdetermined is thus a mean value.

A method which can be used to determine the DAR, herein called DAR UV,consists in measuring spectrophotometrically the ratio of the absorbanceat of a solution of substantially purified conjugate at λ_(D) and 280nm. 280 nm is a wavelength generally used for measuring proteinconcentration, such as antibody concentration. The wavelength λ_(D) isselected so as to allow discriminating the drug from the antibody, i.e.as readily known to the skilled person, λ_(D) is a wavelength at whichthe drug has a high absorbance and λ_(D) is sufficiently remote from 280nm to avoid substantial overlap in the absorbance peaks of the drug andantibody. λ_(D) may be selected as being 252 nm in the case ofmaytansinoid molecules. A method of DAR calculation may be derived fromAntony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies andProtocols, vol 525, 445, Springer Science:

The absorbances for the conjugate at λ_(D) (A_(λD)) and at 280 nm (A₂₈₀)are measured using a classic spectrophotometer apparatus (allowing tocalculate the “DAR parameter”). The absorbances can be expressed asfollows:

A _(λD)=(C _(D)×ε_(DAD))+(C _(A)×ε_(AλD))

A ₂₈₀=(C _(D)×ε_(D280))+(C _(A)×ε_(A280))

wherein:

-   -   C_(D) and C_(A) are respectively the concentrations in the        solution of the drug and of the antibody    -   ε_(DλD) and ε_(D280) are respectively the molar extinction        coefficients of the drug at λ_(D) and 280 nm    -   ε_(AλD) and ε_(A280) are respectively the molar extinction        coefficients of the antibody at λ_(D) and 280 nm.

Resolution of these two equations with two unknowns leads to thefollowing equations:

C _(D)=[(ε_(A280)×_(AλD))−(ε_(AλD) ×A₂₈₀)]/[(ε_(AλD)×ε_(A280))−(ε_(AλD)×ε_(D280))]

C _(A) =[A ₂₈₀−(C _(D)×ε_(D280))]/ε_(A280)

The average DAR is then calculated from the ratio of the drugconcentration to that of the antibody: DAR=C_(D)/C_(A).

In the immunoconjugate according to the invention, the antibody is inparticular specific for human and Macaca fascicularis LAMP1 proteins.

The antibody is in particular a chimeric or humanised antibody. Theantibody may also be an antibody fragment, or a bispecific ormultispecific antibody.

Antibodies binding specifically to human and Macaca fascicularis LAMP1proteins which are particularly contemplated to be included in theimmunoconjugates of the invention are described in further details inthe following “Antibodies” section.

Antibodies

The inventors identified four antibodies (the so-called antibodies“MAb1”, “MAb2”, “MAb3” and “MAb4”) that bind specifically to human LAMP1and distinguish tumoral from non-tumoral tissues. The antibodies MAb1,MAb2, MAb3 allowed for the first time to detect extracellularlyexpressed LAMP1 and thus to perform IHC analysis on Frozen-OCT (fromOptimal Cutting Temperature) specimens and AFA (Alcohol Formalin Aceticacid Fixative) to distinguish cancerous from non-cancerous tissue.

However, IHC analysis of tumor tissues from biobanks or from hospitalsbefore or during patient treatment is routinely done with formalin-fixedparaffin-embedded (FFPE) samples. Although MAb1, MAb2 and MAb3 allowLAMP1 membrane reinforcement in frozen-OCT and AFA (Alcohol FormalinAcetic acid Fixative) sample format, they can not lead to the detectionof LAMP1 reinforcement in FFPE format. One of the reasons is probablythe effect of the formalin fixative combined to the complexity of theprotein. The inventors discovered peptides that allowed the productionof a monoclonal antibody MAb4 that can be furthermore used for IHCexperiments on the FFPE the format and thus allows the application ofthe herein presented methods on FFPE tumor biobanks and FFPE hospitalsamples.

Those four antibodies showed a high binding affinity (within thenanomolar range) to cell surface expressed LAMP1 in cancer cells.Furthermore, at least the anti-LAMP1 MAb1, MAb2 and MAb3 antibodiesshowed a high capacity to trigger internalization of theLAMP1/anti-LAMP1 antibody complex, as shown in example 4.4.

Additionally, the four antibodies are cross-reactive with Macacafascicularis LAMP1 but do not display any cross-reactivity with humanLAMP2 protein.

The binding sites of antibodies MAb1, MAb2 and MAb3 have been mapped toa domain consisting of the first to third loops of human and Macacafascicularis LAMP1 proteins, in particular to the first lumenal domainof human LAMP1. More specifically the binding site of MAb1 was mapped inloops 1-2 and the binding site of MAb2 and MAb3 was mapped in loop1. Theantibodies MAb1 and MAb2 do not compete with each other for binding tohuman LAMP1. Therefore at least two epitopes on LAMP1 have been found tointeract with the antibodies of the invention.

The binding site of Antibody MAb4 has been mapped to a domain consistingof the third to fourth loop of human and Macaca fascicularis LAMP1proteins, in particular to the fourth loop of human LAMP1. Morespecifically the antibody MAb4 binds to a region of Loop 4 comprisingthe amino acids 360 to 375 of human LAMP1 that consists of sequences SEQID NO: 82.

The inventors have determined the sequence of variable heavy and lightchains of these monoclonal antibodies which are directed against thehuman and Macaca fascicularis LAMP1 proteins.

The so-called antibody “MAb1” comprises:

-   -   a variable domain of heavy chain consisting of sequence        QVQLQQSGAELVKPGASVKMSCKASGYIFTNYNIHWVKKSPGQGLEWIGAIYPGNGDAPY        SQKFKDKATLTADKSSSTTYMQLSRLTSEDSAVYYCVRANWDVAFAYWGQGTLVSVSA (SEQ        ID NO: 1, with CDRs shown in bold characters) in which FR1-H        spans amino acid positions 1 to, 25, CDR1-H spans amino acid        positions 26 to 33 (SEQ ID NO: 2), FR2-H spans amino acid        positions 34 to 50, CDR2-H spans amino acid positions 51 to 58        (SEQ ID NO: 3), FR3-H spans amino acid positions 59 to 96,        CDR3-H spans amino acid positions 97 to 107 (SEQ ID NO: 4), and        FR4-H spans amino acid positions 108 to 118, and    -   a variable domain of light chain consisting of sequence        DIQMTQSPPSLSASLGGKVTITCKASQDIDRYMAWYQDKPGKGPRLLIHDTSTLQPGIPSRF        SGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIK    -   (SEQ ID NO: 5, with CDRs shown in bold characters) in which        FR1-L spans amino acid positions 1 to 26, CDR1-L spans amino        acid positions 27 to 32 (SEQ ID NO: 6), FR2-L spans amino acid        positions 33 to 49, CDR2-L spans amino acid positions 50 to 52,        FR3-L spans amino acid positions 53 to 88, CDR3-L spans amino        acid positions 89 to 96 (SEQ ID NO: 7), and FR4-H spans amino        acid positions 97 to 106.

The so-called antibody “MAb2” comprises:

-   -   a variable domain of heavy chain consisting of sequence        QVQLQQSAAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLEWIGYFNPSSGYPE        YNQKFKDKTTLTADKSSNTAFIQLNSLTSEDSAVYYCSRGYYYGSRGYALDFWGQGASVT VSS    -   (SEQ ID NO: 8, with CDRs shown in bold characters) in which        FR1-H spans amino acid positions 1 to 25, CDR1-H spans amino        acid positions 26 to 33 (SEQ ID NO: 9), FR2-H spans amino acid        positions 34 to 50, CDR2-H spans amino acid positions 51 to 58        (SEQ ID NO: 10), FR3-H spans amino acid positions 59 to 96,        CDR3-H spans amino acid positions 97 to 111 (SEQ ID NO: 11), and        FR4-H spans amino acid positions 112 to 122, and    -   a variable domain of light chain consisting of sequence        NIVLTQSPVSLAVSLGQRATISCRASESVDINGNTFMHWYQQKPGQSPKLVIYAASNIESGV        PARFSGSGSSTDFTFTIDPVEADDVATYYCQQFNIEDPWTFGGGTKVEIK    -   (SEQ ID NO: 12, with CDRs shown in bold characters) in which        FR1-L spans amino acid positions 1 to 26, CDR1-L spans amino        acid positions 27 to 36 (SEQ ID NO: 13), FR2-L spans amino acid        positions 37 to 53, CDR2-L spans amino acid positions 54 to 56,        FR3-L spans amino acid positions 57 to 92, CDR3-L spans amino        acid positions 93 to 101 (SEQ ID NO: 14), and FR4-H spans amino        acid positions 102 to 111.

A variant of antibody MAb2, called herein “MAb2_(Can)” was alsogenerated by introducing canonical residues by substitution of A116T inthe variable domain of the heavy chain and by substitution of V9A, V51L,158L, S72G and A108T in the variable domain of the light chain.

The so-called “antibody MAb2_(Can)” comprises:

-   -   a variable domain of heavy chain consisting of sequence    -   QVQLQQSAAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLEWIGYFNPS        SGYPEYNQKFKDKTTLTADKSSNTAFIQLNSLTSEDSAVYYCSRGYYYGSRGYALDFWGQ        GTSVTVSS (SEQ ID NO: 15).    -   a variable domain of light chain consisting of sequence    -   NIVLTQSPASLAVSLGQRATISCRASESVDINGNTFMHWYQQKPGQSPKLLIYAASN        LESGVPARFSGSGSGTDFTFTIDPVEADDVATYYCQQNIEDPWTFGGGTKLEIK (SEQ ID        NO: 16).

Both “MAb2_(Can)” and “MAb2”, under chimeric form, have the sameaffinity for human LAMP1 (see Table 13).

The so-called antibody “MAb3” comprises:

-   -   a variable domain of heavy chain consisting of sequence        QIQLVQSGPELKKPGETVKISCKASGYIFTNYGMNWVKQAPGKGLKWMGWINTYTGESRY        ADDFKGRFALSLETSASTAYLQINNLENEDMATYFCAREDYYGNSPWFFDVWGAGTTVTV SS    -   (SEQ ID NO: 42, with CDRs shown in bold characters) in which        FR1-H spans amino acid positions 1 to 25, CDR1-H spans amino        acid positions 26 to 33 (SEQ ID NO: 43), FR2-H spans amino acid        positions 34 to 50, CDR2-H spans amino acid positions 51 to 58        (SEQ ID NO: 44), FR3-H spans amino acid positions 59 to 96,        CDR3-H spans amino acid positions 97 to 111 (SEQ ID NO: 45), and        FR4-H spans amino acid positions 112 to 122, and    -   a variable domain of light chain consisting of sequence        DIQMTQTTSSLSASLGDRVTISCNASQGINKYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRF        SGSGSGTDYSLTINNLEPEDIATYYCQQYTKLPFTFGSGTKLEIK    -   (SEQ ID NO: 46, with CDRs shown in bold characters) in which        FR1-L spans amino acid positions 1 to 26, CDR1-L spans amino        acid positions 27 to 32 (SEQ ID NO: 47), FR2-L spans amino acid        positions 33 to 49, CDR2-L spans amino acid positions 50 to 52,        FR3-L spans amino acid positions 53 to 88, CDR3-L spans amino        acid positions 89 to 97 (SEQ ID NO: 48), and FR4-H spans amino        acid positions 98 to 107.

A variant of MAb3 (“MAb3 VL_R24_R93”) was generated by introducing intoVL sequence of MAb3 the following amino acid substitutions: N24R andK93R. Accordingly, the variable domain of light chain of MAb3 VL_R24_R93consist of

-   -   DIQMTQTTSSLSASLGDRVTISCRASQGINKYLNWYQQKPDGTVKLLIYYTSTLHSG        VPSRFSGSGSGTDYSLTINNLEPEDIATYYCQQYTRLPFTFGSGTKLEIK (SEQ ID        NO: 51) (the mutated residues as compared with VL of MAb3 being        shown in enlarged characters).

CDR3-L of MAb3 VL_R24_R93 thus consists of QQYTRLPFT (SEQ ID NO: 52).

The so called “MAb4” comprises:

-   -   a variable domain of heavy chain consisting of sequence    -   QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGS        GGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARYRSYDWYFDVWGAGTT        VTVSS (SEQ ID NO: 88, with CDRs shown in bold characters) in        which FR1-H spans amino acid positions 1 to 25, CDR1-H spans        amino acid positions 26 to 33 (SEQ ID NO: 83), FR2-H spans amino        acid positions 34 to 50, CDR2-H spans amino acid positions 51 to        58 (SEQ ID NO: 84), FR3-H spans amino acid positions 59 to 96,        CDR3-H spans amino acid positions 97 to 108 (SEQ ID NO: 85), and        FR4-H spans amino acid positions 109 to 119, and    -   a variable domain of light chain consisting of sequence    -   DIQMTQSPASLSASVGETVTITCRVSGNIHNYLAWYQQKQGKSPQLLVYNAKTLAD        GVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSNPYTFGGGTKLEIK (SEQ ID NO:        89, with CDRs shown in bold characters) in which FR1-L spans        amino acid positions 1 to 26, CDR1-L spans amino acid positions        27 to 32 (SEQ ID NO: 86), FR2-L spans amino acid positions 33 to        49, CDR2-L spans amino acid positions 50 to 52, FR3-L spans        amino acid positions 53 to 88, CDR3-L spans amino acid positions        89 to 97 (SEQ ID NO: 87), and FR4-H spans amino acid positions        98 to 107.

The antibody may also be a humanised antibody or a fragment of ahumanised antibody. For example, the antibody of the invention mayresult from humanisation of any of the antibodies defined above.

Numerous methods for humanisation of an antibody sequence are known inthe art; see e.g. the review by Almagro & Fransson (2008) Front Biosci.13: 1619-1633. One commonly used method is CDR grafting, or antibodyreshaping, which involves grafting of the CDR sequences of a donorantibody, generally a mouse antibody, into the framework scaffold of ahuman antibody of different specificity. Since CDR grafting may reducethe binding specificity and affinity, and thus the biological activityof the parent antibody, back mutations may be introduced at selectedpositions of the CDR grafted antibody in order to retain the bindingspecificity and affinity of the parent antibody. Identification ofpositions for possible back mutations can be performed using informationavailable in the literature and in antibody databases. An alternativehumanization technique to CDR grafting and back mutation is resurfacing,in which non-surface exposed residues of non-human origin are retained,while surface residues are altered to human residues. Anotheralternative technique is known as “guided selection” (Jespers, L. S. etal., 1994, Biotechnology 12(9): 899-993) and can be used to derive froma murine antibody a fully human antibody conserving the epitope andbinding characteristics of the parental antibody. The technique ofhumanization based on molecular dynamic calculations as disclosed in theapplication WO2009/032661 may be used. Thus in one embodiment humanizedantibodies may also be called “resurfaced” antibodies.

For chimeric antibodies, humanisation typically involves modification ofthe framework regions of the variable region sequences.

Amino acid residues that are part of a CDR will typically not be alteredin connection with humanisation, although in certain cases it may bedesirable to alter individual CDR amino acid residues, for example toremove a glycosylation site, a deamidation site, an undesired cysteineresidue, a lysine residue in the case of ADC, N-linked glycosylationoccurs by attachment of an oligosaccharide chain to an asparagineresidue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X maybe any amino acid except Pro. Removal of an N-glycosylation site may beachieved by mutating either the Asn or the Ser/Thr residue to adifferent residue, in particular by way of conservative substitution.Deamidation of asparagine and glutamine residues can occur depending onfactors such as pH and surface exposure. Asparagine residues areparticularly susceptible to deamidation, primarily when present in thesequence Asn-Gly, and to a lesser extent in other dipeptide sequencessuch as Asn-Ala. When such a deamidation site, in particular Asn-Gly, ispresent in a CDR sequence, it may therefore be desirable to remove thesite, typically by conservative substitution to remove one of theimplicated residues. In the case of ADC, attachment of a cytotoxic tomAb could be prepared via covalent linkage to lysine side chain residue.This steric hindrance may interfere with mAb binding to antigen. It maytherefore be desirable to remove the lysine residue, typically by anarginine conservative substitution. Substitution in a CDR sequence toremove one of the implicated residues is also intended to be encompassedby the present invention. The inventors further generated humanizedantibodies “huMAb1_1”, “huMAb1_2”, “huMAb1_3” based on CDR graftingand/or on Molecular Dynamic Trajectories (4D humanization protocol) asdescribed in example 7.2.1 and herein below.

Accordingly, in one embodiment, the anti-LAMP1 antibodies in context ofthe invention are humanized anti-LAMP1 antibodies obtained through CDRgrafting and/or based on Molecular Dynamic Trajectories (4D humanizationprotocol).

Accordingly, in an embodiment, the humanized anti-LAMP1 antibody“huMAb1_1” comprises:

-   -   the variable domain (VH1) of heavy chain consisting of sequence    -   QVQLVQSGAEVKKPGSSVKVSCKASGYIFTNYNIHWVKKSPGQGLEWIGAIYPGNG        DAPYSQKFQGKATLTADTSTSTTYMELSSLRSEDTAVYYCVRANWDVAFAYWGQGTLVTV SS        (SEQ ID NO: 53) and    -   the variable domain (VL1) of light chain of huMAb1_1 consisting        of sequence        DIQMTQSPSSLSASVGDRVTITCKASQDIDRYMAWYQDKPGKAPRLLIHDTSTLQS        GVPSRFSGSGSGRDYTLTISNLEPEDFATYYCLQYDNLWTFGGGTKVEIK (SEQ ID NO:        56).

The humanized antibody “huMAb1_2” comprises:

-   -   a variable domain (VH2) of heavy chain consisting of sequence    -   QVQLVQSGAELVKPGASVKMSCKASGYIFTNYNIHWVKKSPGQGLEWIGAIYPGNG        DAPYSQKFQDRATLTADTSSSTTYMELSSLTSEDSAVYYCVRANWDVAFAYWGQGTLVS VSS        (SEQ ID NO: 54), and    -   a variable domain of light chain (VL2) consisting of sequence    -   DIQMTQSPPSLSASVGGKVTITCKASQDIDRYMAWYQDKPGKGPKLLIHDTSTLQP        GIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIK (SEQ ID NO:        57)

The humanized antibody “huMAb1_3” comprises:

-   -   a variable domain (VH3) of heavy chain consisting of sequence    -   QVQLVQSGAELVKPGASVKMSCKASGYIFTNYNIHWVRQAPGQGLEWIGAIYPGN        GDAPYAQKFQGRATLTADTSSSTTYMELSSLTSEDTAVYYCVRANWDVAFAYWGQGTLV TVSS        (SEQ ID NO: 55), and    -   a variable domain of light chain (VL3) consisting of sequence    -   DIQMTQSPSSLSASVGGKVTITCKASQDIDRYMAWYQQKPGKGPKLLIHDTSTLQP        GVPSRFSGSGSGRDYSLTISSLEPEDIATYYCLQYDNLWTFGGGTKLEIK (SEQ ID        NO:58)

The invention relates to an antibody which binds specifically to humanand Macaca fascicularis LAMP1 proteins.

“Affinity” is defined, in theory, by the equilibrium association betweenthe whole antibody and the antigen. It can be experimentally assessed bya variety of known methods, such as measuring association anddissociation rates with surface plasmon resonance or measuring the EC₅₀in an immunochemical assay (ELISA, FACS). Enzyme-linked immunosorbentassay (ELISA) is a biochemistry assay that uses a solid-phase enzymeimmunoassay to detect the presence of a substance, usually an antigen,in a liquid sample or wet sample. Antigens from the sample are attachedto a surface. Then, a further specific antibody is applied over thesurface so it can bind to the antigen. This antibody is linked to anenzyme, and, in the final step, a substance containing the enzyme'ssubstrate is added. The subsequent reaction produces a detectablesignal, most commonly a color change in the substrate.Fluorescence-activated cell sorting (FACS) provides a method for sortinga heterogeneous mixture of biological cells into two or more containers,one cell at a time, based upon the specific light scattering andfluorescent characteristics of each cell. In these assays, the EC₅₀ isthe concentration of the antibody which induces a response halfwaybetween the baseline and maximum after some specified exposure time on adefined concentration of antigen by ELISA (enzyme-linked immuno-sorbentassay) or cell expressing the antigen by FACS (Fluorescence ActivatedCell Sorting).

A monoclonal antibody binding to antigen 1(Ag1) is “cross-reactive” toantigen 2 (Ag2) when the EC₅₀s are in a similar range for both antigens.In the present application, a monoclonal antibody binding to Ag1 iscross-reactive to Ag2 when the ratio of affinity of Ag2 to affinity ofAg1 is equal or less than 10 (in particular 5, 2, 1 or 0.5), affinitiesbeing measured with the same method for both antigens.

A monoclonal antibody binding to Ag1 is “not significantlycross-reactive” to Ag2 when the affinities are very different for thetwo antigens. Affinity for Ag2 may not be measurable if the bindingresponse is too low. In the present application, a monoclonal antibodybinding to Ag1 is not significantly cross-reactive to Ag2, when thebinding response of the monoclonal antibody to Ag2 is less than 5% ofthe binding response of the same monoclonal antibody to Ag1 in the sameexperimental setting and at the same antibody concentration. Inpractice, the antibody concentration used can be the EC₅₀ or theconcentration required to reach the saturation plateau obtained withAg1.

A monoclonal antibody “binds specifically” to Ag1 when it is notsignificantly cross-reactive to Ag2.

The antibody according to the invention binds specifically to human andMacaca fascicularis LAMP1 proteins. It does not significantlycross-react with human LAMP2 (SEQ ID NO: 40).

In one embodiment, the antibody according to the invention has anaffinity (EC₅₀) for human and/or cynomolgus monkey LAMP1 expressed atthe cell surface of a recombinant cell line, wherein the cell line maybe HEK293 and/or HCT116 and the apparent affinity measured via FlowCytometry is ≤70 nM, for example ≤60 nM, ≤50 nM, ≤45 nM, ≤40 nM, ≤35 nM,≤30 nM, ≤25 nM, ≤20 nM, ≤15 nM or ≤10 nM.

In one embodiment, the antibody according to the invention has anaffinity (EC₅₀) for full length human and cynomolgus monkey LAMP1expressed at the cell surface of a recombinant cell line, wherein thecell line may be HCT116 and the apparent affinity measured via FlowCytometry is ≤20 nM, in particular ≤10 nM, ≤8 nM or ≤7 nM.

In one example, the antibody according to the invention has an affinity(EC₅₀) for cynomolgus monkey LAMP1 expressed at the cell surface of arecombinant cell line, wherein the cell line may be HEK293 and theapparent affinity measured via Flow Cytometry is ≤50 nM, for example ≤40nM or ≤35 nM.

In another example, the antibody according to the invention has a KD forfull purified human LAMP1 (SEQ ID NO 28) expressed in HEK293 cellsmeasured via surface plasmon resonance (SPR) is 70 nM, for example 60nM, 50 nM, 40 nM, 30 nM, 20 nM or 10 nM.

The use of surface plasmon resonance to determine is known to theskilled in the art. In one example the binding kinetics of for examplethe murine, chimer or humanized anti-LAMP1 mAbs were determined bysurface plasmon resonance assay using typically a BIAcore 2000 (BIAcoreInc., Uppsala, N.J.). Therefore, for example a CM5 BIAcore biosensorchip was docked into the instrument and activated with for example 70 μLof 1:1 NHS/EDC at room temperature. Typically, a mouse anti-αhuman FcIgG1 (BIAcore #BR-1008-39) and rabbit anti-αmurine Fc IgG1 (BIAcore#BR-1008-38) (50 μg/mL in 1 M acetate buffer, pH5) were immobilized onthe activated chips in all flow cells. The immobilization was carriedout at a flow rate of for example 10 μL/min up to saturation. The chipwas then blocked by for example injection of 70 μL of ethanolamine-HCl,pH 8.5, followed by one wash with 3 M MgCl₂ for anti-αhuman Fc IgG1 andone wash with 10 mM Glycine-HCl pH 1.7 for anti-αmurine Fc IgG1. Tomeasure the binding of for example anti-LAMP1 mAbs to LAMP1, antibodieswere used at 1-5 μg/mL in BIAcore running buffer (HBS-EP). The antigenfor example (Sequence ID No 28 protein produced as described in example6.2) was injected from for example 1 to 256 nM. Following completion ofthe injection phase, dissociation was monitored in a BIAcore runningbuffer at the same flow rate for typically 600 sec. The surface wastypically regenerated between injections using for example 2×5 μL 3 MMgCl₂ (2×30 s) or anti-αhuman Fc IgG1 and 1×30 μL 10 mM Glycine-HCl pH1.7 for anti-αmurine Fc IgG1 (180 s). Individual sensorgrams weretypically analyzed using BIAevaluation software.

Thus, the polypeptide according to the invention may be used intoxicological studies performed in monkeys, wherein the toxicity profileobtained from those studies is relevant to anticipate potential adverseeffects in humans.

Alternatively, or furthermore, the antibody according to the inventionhas an affinity (EC₅₀) for LAMP1 expressed on the surface of advancedhuman primary colon tumor CR-IGR-034P and measured via Flow Cytometry is≤50 nM, ≤40 nM, in particular ≤30 nM, ≤20 nM or 5 nM.

Antibody binding capacity or ABC is the quantification of cell surfaceantigen. ABC can be measured using QIFIKIT® (Registered trademark ofBIOCYTEX). The antibodies according to the invention have a high ABC onmany Patient derived Xenografts of different origin (≥20.000, inparticular ≥50.000, ≥100.000, ≥150.000 ABC) and tumor cell lines, inparticular colon tumor cells such as Colo205, SW480 or LS174T (≥1.500,≥2.500, ≥4.000 ABC).

Alternatively, or furthermore, the antibody according to the inventionhas the ability to internalize and recycle LAMP1 to the cell membrane.In particular, when bound by an antibody according to the invention, amolecule of LAMP1 at the membrane of cancer cell has the capacityundergo at least 1, 4, 7 or 9 recyclings at the cell membrane. In otherwords, one molecule of LAMP1 expressed at the surface of a cancer cellcan be bound by, and therefore internalize, at least 2, 5, 8 or 10molecules of antibody according to the invention. Still in other words,according to an embodiment, at least 2, 5, 8 or 10 molecules of antibodyaccording to the invention are internalized by one molecule of LAMP1expressed at the surface of a cancer cell.

Internalization may be assayed for instance by determining aninternalization score or by a fluorescence-based quenching method.

The internalization score (IS) is defined as a ratio of the fluorescenceintensity inside the cell to the intensity of the entire cell. It may bemeasured as described by using the imaging flow cytemeterImageStream^(x) (from the supplier Amnis® Corporation, 2505 ThirdAvenue, Suite 210, Seattle, Wash. 98121-1480, www.amnis.com). The higherthe score, the greater the fluorescence intensity is inside the cell. Asdescribed by Amnis (see www.amnis.com), the inside of the cell isdefined by an erosion of a mask that fits the membrane of the cell. Thescore is invariant to cell size and can accommodate concentrated brightregions and small dim spots. The ratio is mapped to a logarithmic scaleto increase the dynamic range to values between {−inf, inf}. Thethickness of the membrane (in pixels) determines which pixels are usedto define the boundary and the membrane portions of the cell. The usersupplies an ‘internal’ mask based on the brightfield image that coversthe inside of the cell, the thickness of the membrane in pixels and thefluorescent channel of interest. The cell is divided into 2 regions:External (B) and internal (I). The user supplies the internal region asthe mask. The external region is determined by: 1. Dilating the internalmask by the membrane thickness. 2. Combining 1 with the object mask ofthe channel of interest. 3. External region equals mask 2 and not theinternal mask. Next, the mean intensity of the upper quartile of thepixels in each region is determined. The Internalization Score (IS) isthen computed as follows:

${{I\; S} = {\log \left( \frac{a}{1 - a} \right)}},{{{where}\mspace{14mu} a} = {\frac{m_{I}}{m_{I} + m_{B}}\frac{p_{I}}{P_{B}}}}$

-   -   mI=Mean intensity of upper quartile pixels in I, mB=Mean        intensity of upper quartile pixels in B,    -   pI=Peak intensity of upper quartile pixels in I, pB=Peak        intensity of upper quartile pixels in B.

In the case of transferrin, (Williams A. et al., 1996, Biomembranes,4:255-287) the authors have obtained an IS of 0 when the cells were lefton ice and an IS of 0.9 when the cells were incubated at 37° C. for onehour.

For the antibodies of the invention, the inventors have shown that theinternalization scores (IS) at 37° C. were 10-fold higher than at 4° C.Since internalization of antibodies does not take place at 4° C., theinternalization scores obtained at 4° C. reflect the density of LAMP1molecules at the cell surface. A 10-fold higher value of the ISparameter at 37° C. than at 4° C. therefore means that the LAMP1 proteinis quickly and repeatedly recycling at the cell membrane. In other wordsthe antibodies according to the invention have a very highinternalization capacity, much higher than the capacity calculated fromthe density of the LAMP1 protein at the cell surface.

Quantification of internalization can also be performed byfluorescence-based quenching methods. In particular, afluorescence-based Alexa488-quenching method has been described toanalyze internalisation of targeting agents (Frejd et al. 2010,International Journal of Oncology, 36: 757-763). According to saiddescription, internalization is calculated as the Mean fluorescenceintensity (MFI) value of quenched cells (intracellular compartmentsonly) divided by the MFI value of unquenched cells (both cell surfaceand intracellular compartments) at 37° C., according to the followingformula:

${Percentage}\mspace{14mu} {of}\mspace{14mu} {internalized}\mspace{14mu} {fraction} \text{:}\; \frac{{FL}\mspace{14mu} {of}\mspace{14mu} {quenched}\mspace{14mu} {cells}\mspace{14mu} {at}\mspace{14mu} 37{^\circ}\mspace{14mu} {C.}}{{FL}\mspace{14mu} {of}\mspace{14mu} {unquenched}\mspace{14mu} {cells}\mspace{14mu} {at}\mspace{14mu} 37{^\circ}\mspace{14mu} {C.}} \times 100$

Cells incubated with Alexa488-labelled compounds at 4° C. are used as acontrol since internalization of antibodies does not take place at 4° C.

The inventors showed that after quenching, the total fluorescence ofAlexa488-MAb1 measured from cells labelled at 37° C. (both cell surfaceand intracellular compartments) was 10-fold higher than the fluorescenceof cells labelled at 4° C. (cell surface) after 4 h (example 4.4).Accordingly, these results also indicate that the LAMP1 protein isquickly and repeatedly recycling at cell membrane.

Thus, the inventors showed for the first time that LAMP1 can function asa receptor mediating the internalization of antibodies and suggest thatavailability of specific internalizing antibodies should aid indeveloping novel therapeutic methods to target toxins, drugs orshort-range isotopes to be delivered specifically to the interior of thecancer cells.

Furthermore the inventors could show that the results from example 4.4taken together indicate that each LAMP1 molecule is involved in several(at least up to 10 on average) internalization cycles via recycling atcell membrane during the course of the experiment.

The antibody of the invention binds specifically to a domain consistingin particular of the first to third loops of human and Macacafascicularis LAMP1 proteins. The domain consisting of the first to thirdloops of human LAMP1 protein is defined by the amino acids Ala29 toIle309 of SEQ ID NO: 24, and the domain consisting of the first to thirdloops of Macaca fascicularis LAMP1 protein is defined by the amino acidsAla27 to Thr307 of SEQ ID NO: 39. According to an embodiment, theantibody of the invention binds specifically to the first lumenal domainof human and Macaca fascicularis LAMP1 proteins.

The first lumenal domain of human LAMP1 is defined by the amino acids atpositions Ala29 to Arg195 of SEQ ID NO: 24, and the first lumenal domainof Macaca fascicularis LAMP1 protein is defined by the amino acids atpositions Ala27 to Arg193 of SEQ ID NO: 39. More specifically, theantibody can bind to the human and Macaca fascicularis first lumenaldomain indifferently whether expressed as a soluble extracellular domain(e.g. amino acids Ala29-Met382 for human LAMP1 (SEQ ID NO: 24) orAla27-Met380 for Macaca fascicularis LAMP1 (SEQ ID NO: 39)), or as amembrane-anchored full-length LAMP1 protein recombinantly expressed atthe surface of a cell line, for instance HT29, Colo205 and HCT116,HEK293 cell line. The inventors demonstrated that MAb1 binds to theamino acids 101 to 195 of SEQ ID NO: 24 corresponding to Loop 2 of humanLAMP1, for example to the amino acids 101 to 110 (SEQ ID NO: 72), 144 to157 (SEQ ID NO:73) and 174 to 188 (SEQ ID NO: 74) of SEQ ID NO: 24 asherein described in example 4.8. It has been further identified bycrystallography, that the binding site of MAb1 further encompasses theamino acids Asn35, Cys80, Gly 81, Glu83, Asn84 located in loop 1 of SEQID NO: 24.

Accordingly, MAb1 also binds to the amino acids at positions 29 to 100of SEQ ID NO: 24 corresponding to Loop 1 of human LAMP1, for example toa region that consists of the amino acids at positions 35 to 84 of SEQID NO: 24 (SEQ ID NO:97), or to two regions that consists of Asn35 ofSEQ ID NO: 24 and amino acids at positions 80-84 of SEQ ID NO: 24.

Furthermore both MAb2 and MAb3 bind to the amino acids 29 to 100 (SEQ IDNO: 77) of SEQ ID NO: 24 corresponding to Loop 1 of human LAMP1, forinstance MAb2 and MAb3 both bind to the amino acids 29 to 41 (SEQ ID NO:75) and 68 to 80 (SEQ ID NO: 76) of SEQ ID NO: 24.

In a further antibody, the antibody of the invention binds specificallyto the second luminal domain of human and Macaca fascicularis LAMP1proteins, for instance to the fourth loop.

The fourth loop of human LAMP1 protein consists of amino acids atpositions Leu310 to Met382 of SEQ ID NO: 24 and the fourth loop ofMacaca fascicularis LAMP1 protein consists of amino acids at positionsLeu 308 to Met380 of SEQ ID NO: 39.

More specifically, the antibody binds to a region of Loop 4 comprisingthe amino acids 360 to 375 of human LAMP1 that consists of sequences SEQID NO: 82.

In another embodiment, the antibody of the invention binds specificallyto human and Macaca fascicularis LAMP1 proteins indifferently whether innon-glycosylated or glycosylated form.

Accordingly, in an embodiment, the invention relates to an antibodywhich binds to:

-   -   three regions of Loop 2 of human LAMP1 that consist of sequences        SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, respectively,        and optionally further to a region of Loop1 of human LAMP1 that        consists of sequence (SEQ ID NO:97); or    -   two regions of Loop 1 of human LAMP1 that consist of sequences        SEQ ID NO: 75 and SEQ ID NO: 76, respectively; or    -   a region of Loop4 of human LAMP1 that consist of sequence SEQ ID        NO: 82.

Furthermore, the inventors identified the residues R146, D150, K152,R106, A108, N181, S182, S183, R186 and G187 of SEQ ID NO: 24 as likelyto interact with MAb1 as described in example 6.5. They furtheridentified the residues A29, M30, M32, G36, A40, S69, D70, T72, V74,L75, and R77 of SEQ ID NO: 24 as likely to interact with MAb2 and/orMAb3. Those residues have been individually replaced by an alanineresidue in the LAMP1 sequence derived from hLAMP1_ΔGYQTI and encoded inplasmid pXL5626 as described in example 6.6. The inventors observed lossof binding to MAb1 for alanine mutations at positions I149, D150 andR186 of SEQ ID NO: 24 in the LAMP1 protein, indicating that thesepositions are important for MAb1 binding to LAMP1. Furthermore, loss ofbinding was demonstrated for LAMP1 to MAb3 for alanine mutations atpositions G38 and D70 of SEQ ID NO: 24 due to Ala substitution in LAMP1protein indicating that these positions are important for MAb3 bindingto LAMP1.

Accordingly, in an embodiment, the invention relates to an antibodywhich binds to:

-   -   the amino acids 1149, D150 and R186 of SEQ ID NO: 24, or    -   the amino acids G38 and D70 of SEQ ID NO: 24, or

The invention also provides for an antibody which competes for bindingto a domain consisting of the first to third loops of human and Macacafascicularis LAMP1 proteins with an antibody selected from the groupconsisting of the so-called antibodies Mab1, Mab2, MAb2_(Can), MAb3,MAb3 VL_R24_R93, huMAb1_1 and huMAb1_2, huMAb1_3 i.e.:

-   -   (i) an antibody comprising a variable domain of heavy chain of        sequence SEQ ID NO: 1 and/or a variable domain of light chain of        sequence of sequence SEQ ID NO: 5; or    -   (ii) an antibody comprising a variable domain of heavy chain of        sequence SEQ ID NO: 8 and/or a variable domain of light chain of        sequence of sequence SEQ ID NO: 12; or    -   (iii) an antibody comprising a variable domain of heavy chain of        sequence SEQ ID NO: 15 and/or a variable domain of light chain        of sequence of sequence SEQ ID NO: 16; or    -   (iv) an antibody comprising a variable domain of heavy chain of        sequence SEQ ID NO: 42 and/or a variable domain of light chain        of sequence of sequence SEQ ID NO: 46; or    -   (v) an antibody comprising a variable domain of heavy chain of        sequence SEQ ID NO: 42 and/or a variable domain of light chain        of sequence of sequence SEQ ID NO: 51; or    -   (vi) an antibody comprising a variable domain of heavy chain of        sequence SEQ ID NO: 53 and/or a variable domain of light chain        of sequence of sequence SEQ ID NO: 56; or    -   (vii) an antibody comprising a variable domain of heavy chain of        sequence SEQ ID NO: 54 and/or a variable domain of light chain        of sequence of sequence SEQ ID NO: 57; or    -   (viii) an antibody comprising a variable domain of heavy chain        of sequence SEQ ID NO: 55 and/or a variable domain of light        chain of sequence of sequence SEQ ID NO: 58.

In an embodiment, said antibody competes for binding to the firstlumenal domain of human and Macaca fascicularis LAMP1 proteins. Forinstance the invention provides for an antibody which competes forbinding to:

-   -   three regions of Loop 2 of human LAMP1 that consist of sequences        SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, respectively; or    -   two regions of Loop 1 of human LAMP1 that consist of sequences        SEQ ID NO:75 and SEQ ID NO: 76, respectively,    -   with an antibody comprising a variable domain of heavy chain and        a variable domain of light chain as defined according to i-viii)        above, as appropriate (i.e. with said three regions of Loop 2        for an antibody as defined according to i and vi-viii) above, or        with said two regions of Loop 1 for an antibody as defined        according to ii-v)).

In one embodiment the competition is determined by use of an ELISA asdescribed in Example 4.8 of the specification, wherein competition isdefined by a signal of less than 80% of signal compared to mAb controlalone as assessed by absorption, when the two competing antibodies arein solution at similar molarity, and wherein competition is defined by asignal of less than 80%, for instance less than_70%, 60%, 50%, 40%, 30%,20%, 10%.

The ability of an antibody to compete for binding to a domain consistingof the first to third loops, in particular to the first lumenal domain,of human and Macaca fascicularis LAMP1 proteins with an antibodycomprising the variable heavy and light chains of an antibody selectedfrom the group consisting of the so-called antibodies MAb1, MAb2,MAb2_(Can), MAb3, MAb3_VLR24-R93, huMAb1_1, huMAb1_2 and huMAb1_3(hereafter a “reference” antibody) may be readily assayed, for instance,by competitive ELISA wherein the antigen (i.e. a polypeptide comprisingor consisting of a fragment of human or Macaca fascicularis LAMP1including the first to third loops of LAMP1, or the first lumenaldomain, in particular a protein containing the first lumenal domain ofLAMP1 from human and cynomolgus origin such as presented in example 6.3)is bound to a solid support and two solutions containing the candidateantibody and the reference antibody, respectively, are added and theantibodies are allowed to compete for binding to the antigen. The amountof reference antibody bound to the antigen may then be measured, andcompared to the amount of reference antibody bound to the antigen whenmeasured against a negative control. An amount of bound referenceantibody in presence of the candidate antibody decreased as compared tothe amount of bound reference antibody in presence of the negativecontrol indicates that the candidate antibody has competed with thereference antibody. Conveniently, the reference antibody may be labeled(e.g. fluorescently) to facilitate detection of bound referenceantibody. Repeated measurements may be performed with serial dilutionsof the candidate and/or reference antibody.

In another example binding competition between MAb1 and MAb2 or MAb3 canbe typically measured between two anti-LAMP1 mAbs by ELISA withrecombinant human LAMP1 coated on plate (as described in example 6.2).Briefly, typically two mAbs were added simultaneously at concentrationsof for example 0.06 and 15 mg/L, the concentration of typically 0.06mg/L being close to the EC₅₀. MAb format was chosen so that the two mAbshad different Fc domains (either human or murine). Individualmeasurements of mAb binding could be performed typically by their uniquespecific binding to Fc (for example with Peroxidase-AffiniPure GoatAnti-Human IgG Ab, Fcγ Fragment Specific (Jackson 109-035-098) or withPeroxidase-AffiniPure Goat Anti-Mouse IgG Ab, Fcγ Fragment Specific(Jackson 115-035-164)). The results were reported as a percentage of thevalue obtained from the mAb alone at the same concentration.

In particular, the antibody according to the invention comprises the CDRsequences of the heavy and/or light chains of one of so-calledanti-LAMP1 antibodies MAb1, MAb2 and MAb3. More specifically, theantibody can comprise the CDR sequences of the heavy light chain, or theCDR sequences of the heavy and light chains, of one of so-calledanti-LAMP1 antibodies MAb1, MAb2, MAb3 and MAb3 VL_R24_R93.

Accordingly, the antibody of the invention may comprise:

-   -   (i) a CDR1-H of sequence SEQ ID NO: 2 or a sequence differing        from SEQ ID NO: 2 by one amino acid substitution, a CDR2-H of        sequence SEQ ID NO: 3 or a sequence differing from SEQ ID NO: 3        by one amino acid substitution, and a CDR3-H of sequence SEQ ID        NO: 4 or a sequence differing from SEQ ID NO: 4 by one amino        acid substitution; and/or        a CDR1-L of sequence SEQ ID NO: 6 or a sequence differing from        SEQ ID NO: 6 by one amino acid substitution, a CDR2-L of        sequence DTS or a sequence differing from DTS by one amino acid        substitution and a CDR3-L of sequence SEQ ID NO: 7 or a sequence        differing from SEQ ID NO: 7 by one amino acid substitution; or    -   (ii) a CDR1-H of sequence SEQ ID NO: 9 or a sequence differing        from SEQ ID NO: 9 by one amino acid substitution, a CDR2-H of        sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO:        10 by one amino acid substitution, a CDR3-H of sequence SEQ ID        NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino        acid substitution; and/or        a CDR1-L of sequence SEQ ID NO: 13 or a sequence differing from        SEQ ID NO: 13 by one amino acid substitution, a CDR2-L of        sequence AAS or a sequence differing from AAS by one amino acid        substitution, and a CDR3-L of sequence SEQ ID NO: 14 or a        sequence differing from SEQ ID NO: 14 by one amino acid        substitution; or    -   (iii) a CDR1-H of sequence SEQ ID NO: 43 or a sequence differing        from SEQ ID NO: 43 by one amino acid substitution, a CDR2-H of        sequence SEQ ID NO: 44 or a sequence differing from SEQ ID NO:        44 by one amino acid substitution, and a CDR3-H of sequence SEQ        ID NO: 45 or a sequence differing from SEQ ID NO: 45 by one        amino acid substitution; and/or        a CDR1-L of sequence SEQ ID NO: 49 or a sequence differing from        SEQ ID NO: 47 by one amino acid substitution, a CDR2-L of        sequence YTS or a sequence differing from YTS by one amino acid        substitution, and a CDR3-L of sequence SEQ ID NO: 48 or SEQ ID        NO: 52 or a sequence differing from SEQ ID NO: 48 or SEQ ID NO:        52 by one amino acid substitution.

In a further embodiment, the antibody according to the inventioncomprises the CDR sequences of the heavy and/or light chains ofso-called anti-LAMP1 antibody MAb4. More specifically, the antibody cancomprise the CDR sequences of the heavy light chain, or the CDRsequences of the heavy and light chains, of the so-called anti-LAMP1antibody MAb4.

Accordingly, the antibody of the invention may comprise a CDR1-H ofsequence SEQ ID NO: 83, a CDR2-H of sequence SEQ ID NO: 84, a CDR3-H ofsequence SEQ ID NO: 85, a CDR1-L of sequence SEQ ID NO: 86, a CDR2-L ofsequence NAK, and a CDR3-L of sequence SEQ ID NO: 87.

Furthermore, the antibody of the invention may comprise, or consist of,a heavy chain of sequence SEQ ID NO: 98 and/or a light chain of sequenceof sequence SEQ ID NO: 99 (i.e heavy and/or light chain of MAb4 asdescribed in example 17.2.3).

In one embodiment this antibody may be chimeric, humanized, or anantibody fragment.

In the antibody of the invention, one individual amino acid may bealtered by substitution, in particular by conservative substitution, inone or more (in particular in only one) of the above CDR sequences. Suchan alteration may be intended for example to remove a glycosylation siteor a deamidation site, in connection with humanisation of the antibody.Another alteration could also be intended to remove a lysine in a CDR,since covalent attachment to cytotoxic via lysine side chain residue mayinterfere with binding to antigen in the case of ADC. For instance, SEQID NO: 48 and SEQ ID NO: 52 are CDR3-L sequences that differ by oneamino acid substitution at their position 5.

According to an embodiment, the antibody comprises

-   -   (i) a CDR1-H of sequence SEQ ID NO: 2, a CDR2-H of sequence SEQ        ID NO: 3, and a CDR3-H of sequence SEQ ID NO: 4; and/or a CDR1-L        of sequence SEQ ID NO: 6, a CDR2-L of sequence DTS, and a CDR3-L        of sequence SEQ ID NO: 7; or    -   (ii) a CDR1-H of sequence SEQ ID NO: 9, a CDR2-H of sequence SEQ        ID NO: 10, a CDR3-H of sequence SEQ ID NO: 11; and/or a CDR1-L        of sequence SEQ ID NO: 13, a CDR2-L of sequence AAS, and a        CDR3-L of sequence SEQ ID NO: 14; or    -   (iii) a CDR1-H of sequence SEQ ID NO: 43, a CDR2-H of sequence        SEQ ID NO: 44, and a CDR3-H of sequence SEQ ID NO: 45, and/or a        CDR1-L of sequence SEQ ID NO: 47, a CDR2-L of sequence YTS, and        a CDR3-L of sequence SEQ ID NO: 48 or SEQ ID NO: 52.

In particular, the antibody can comprise:

-   -   (i) a CDR1-H of sequence SEQ ID NO: 2, a CDR2-H of sequence SEQ        ID NO: 3, a CDR3-H of sequence SEQ ID NO: 4, and/or a CDR1-L of        sequence SEQ ID NO: 6, a CDR2-L of sequence DTS, and a CDR3-L of        sequence SEQ ID NO: 7; or    -   (ii) a CDR1-H of sequence SEQ ID NO: 9, a CDR2-H of sequence SEQ        ID NO: 10, a CDR3-H of sequence SEQ ID NO: 11, and/or    -   a CDR1-L of sequence SEQ ID NO: 13, a CDR2-L of sequence AAS,        and a CDR3-L of sequence SEQ ID NO: 14; or    -   (iii) a CDR1-H of sequence SEQ ID NO: 43, a CDR2-H of sequence        SEQ ID NO: 44, a CDR3-H of sequence SEQ ID NO: 45, and/or    -   CDR1-L of sequence SEQ ID NO: 47, a CDR2-L of sequence YTS, and        a CDR3-L of sequence SEQ ID NO: 48 or SEQ ID NO: 52, or    -   (iv) a fragment of an antibody as defined in (i), (ii), or        (iii).

The inventors cristallized recombinant Fab from huMAb1_1 that wasidentified to bind to loop 1 and loop 2 in a complex withnon-glycosylated LAMP1 protein according to the protocol described inexample 7.3.1. Based on the determination of the tridimensionalstructure of huMab1_1 in complex with LAMP1, most of its CDRs can beassociated to specific canonical structure as referenced in Al-Lazikini,Lesk and Chothia (1997) J. Mol. Biol. 273:927-948 mentioned above. Thecristall structure allowed determining mutations that can be introducedinto the CDRS without disturbing said canonical structure. It is knownto the skilled in the art that disturbation of said canonical structurewould result in a modified binding behavior. They thus identified byanalyzing the crystallographic structure, that Q27 and D28 of SEQ ID NO:68 located in CDR1-L can be replaced by any amino acid as long as theloop retains the canonical structure κ2B and I129 of SEQ ID NO: 68 canbe replaced by an equivalent hydrophobic residues, for instance Leu orVal. T51 of SEQ ID NO: 68 and S52 of SEQ ID NO: 68, both located inCDR2-L can be replaced by a Ser, in case of T51 and by any amino acid,in the case of S52, as long as this loop retains the classic γ-turnconformation. Residues D92, N93, L94 of SEQ ID NO: 68, located in CDR3-Lcan be replaced by any amino acids as long as the loop retains canonicalstructure λ1B. Furthermore, G26 of SEQ ID NO: 69, located in CDR-1H canbe replaced by any amino acid, Y27 of SEQ ID NO: 69, located in CDR-1Hby a phenylalanine, T30 of SEQ ID NO: 69, located in CDR-1H by any aminoacid, as long as the loop retains the canonical structure 1. ResiduesD102, V103 and A104 of SEQ ID NO: 69, located in CDR-3H can be replacedby any amino acid of similar sizes and properties.

Accordingly, the invention provides for an antibody which binds to threeregions of Loop 2 of human LAMP1 that consist of sequences SEQ ID NO:72, SEQ ID NO: 73 and SEQ ID NO: 74, respectively; and comprises

-   -   a) a CDR1-L consisting of sequence X₁X₂X₃DRY (SEQ ID NO:93)        wherein each of X₁ and X₂ is any amino acid and X₃ is selected        from Ile, Leu and Val; and    -   a CDR2-L consisting of sequence DX₁X₂ wherein X₁a is selected        from T or S and X₂ is any amino acid; and    -   a CDR3-L consisting of the sequence LQYX₁X₂X₃WT, in which X₁, X₂        and X₃ is any amino acid; and/or    -   b) a CDR1-H consisting of sequence X₁X₂IFX₃NYN (SEQ ID NO: 82)        wherein each of X₁ and X₃ are any amino acid and X₂ is selected        from Tyr or Phe; and a CDR2-H consisting of SEQ ID NO: 3; and        -   CDR3-H consisting of sequence VRANWX₁X₂X₃FAY (SEQ ID NO: 84)            wherein each of X₁, X₂, X₃, is any amino acid.

In one embodiment said antibody retains the ability to bind to loop 2.

The skilled in the art knows methods to verify if the antibody accordingto the definition retains its ability to bind to three regions of loop 2of human LAMP1 that consist of sequences SEQ ID NO: 72, SEQ ID NO: 73and SEQ ID NO: 74, respectively, and thus does not suffer from adisturbed canonical structure.

The invention also provides antibodies as defined above furthercomprising at least the variable domain of heavy chain and/or thevariable domain of light chain of one of the so-called anti-LAMP1antibodies MAb1, MAb2, MAb2_(Can), MAb3, MAb3 VL_R24_R93, huMAb1_1 andhuMAb1_2, huMAb1_3, for instance MAb1, MAb2, MAb2_(Can), MAb3, MAb3VL_R24_R93.

Thus the invention relates in particular to an antibody which comprises:

-   -   (i) a variable domain of heavy chain of sequence SEQ ID NO: 1 or        a sequence at least 85% identical thereto and/or a variable        domain of light chain of sequence of sequence SEQ ID NO: 5, or a        sequence at least 85% identical thereto; or    -   (ii) a variable domain of heavy chain of sequence SEQ ID NO: 8,        or a sequence at least 85% identical thereto, and/or a variable        domain of light chain of sequence of sequence SEQ ID NO: 12, or        a sequence at least 85% identical thereto; or    -   (iii) a variable domain of heavy chain of sequence SEQ ID NO:        15, or a sequence at least 85% identical thereto, and/or a        variable domain of light chain of sequence of sequence SEQ ID        NO: 16, or a sequence at least 85% identical thereto; or    -   (iv) a variable domain of heavy chain of sequence SEQ ID NO: 42,        or a sequence at least 85% identical thereto, and/or a variable        domain of light chain of sequence of sequence SEQ ID NO: 46 or        SEQ ID NO: 51, or a sequence at least 85% identical thereto; or    -   (v) a variable domain of heavy chain of sequence SEQ ID NO: 53        or a sequence at least 85% identical thereto and/or a variable        domain of light chain of sequence of sequence SEQ ID NO: 56, or        a sequence at least 85% identical thereto; or    -   (vi) a variable domain of heavy chain of sequence SEQ ID NO: 54        or a sequence at least 85% identical thereto and/or a variable        domain of light chain of sequence of sequence SEQ ID NO: 57, or        a sequence at least 85% identical thereto; or    -   (vii) a variable domain of heavy chain of sequence SEQ ID NO: 55        or a sequence at least 85% identical thereto and/or a variable        domain of light chain of sequence of sequence SEQ ID NO: 58, or        a sequence at least 85% identical thereto.

In one embodiment, the invention relates to an isolated anti-LAMP-1antibody which comprises:

-   -   (i) a CDR1-H of sequence SEQ ID NO: 2, a CDR2-H of sequence SEQ        ID NO: 3, and a CDR3-H of sequence SEQ ID NO: 4; and/or a CDR1-L        of sequence SEQ ID NO: 6, a CDR2-L of sequence DTS, and a CDR3-L        of sequence SEQ ID NO: 7; or    -   (ii) a CDR1-H of sequence SEQ ID NO: 9, a CDR2-H of sequence SEQ        ID NO: 10, a CDR3-H of sequence SEQ ID NO: 11; and/or a CDR1-L        of sequence SEQ ID NO: 13, a CDR2-L of sequence AAS, and a        CDR3-L of sequence SEQ ID NO: 14; or    -   (iii) a CDR1-H of sequence SEQ ID NO: 43, a CDR2-H of sequence        SEQ ID NO: 44, and a CDR3-H of sequence SEQ ID NO: 45, and/or a        CDR1-L of sequence SEQ ID NO: 47, a CDR2-L of sequence YTS, and        a CDR3-L of sequence SEQ ID NO: 48 or SEQ ID NO: 52; or    -   (iv) CDR1-H of sequence SEQ ID NO: 83, a CDR2-H of sequence SEQ        ID NO: 84, a CDR3-H of sequence SEQ ID NO: 85, and/or a CDR1-L        of sequence SEQ ID NO: 86, a CDR2-L of sequence NAK, and a        CDR3-L of sequence SEQ ID NO: 87; or a heavy chain of sequence        SEQ ID NO: 60 or a light chain of sequence SEQ ID NO: 59; or    -   (v) a heavy chain of sequence SEQ ID NO: 62 or a light chain of        sequence SEQ ID NO: 61; or    -   (vi) a heavy chain of sequence SEQ ID NO: 64 or a light chain of        sequence SEQ ID NO: 63.

For instance, the sequence of the variable domain of heavy or lightchain may differ from the reference sequence SEQ ID NO: 1, 5, 8, 12, 15,16, 42, 46 or 51, 53, 56, 54, 57, 55, 58, for instance from SEQ ID NO:1, 5, 8, 12, 15, 16, 42, 46 or 51 as appropriate, by one or more aminoacid substitution(s), in particular by one or more conservative aminoacid substitution(s) and/or substitution(s) with canonical residues.

In particular, the sequence of the variable domain of heavy or lightchain may differ from the reference sequence SEQ ID NO: 1, 5, 8, 12, 15,16, 42, 46 or 51, 53, 56, 54, 57, 55, 58, for example from SEQ ID NO: 1,5, 8, 12, 15, 16, 42, 46 or 51 by conservative amino acidsubstitution(s), only.

The sequence alterations as compared with sequence SEQ ID NO: 1, 5, 8,12, 15, 16, 42, 46 or 51, 53, 56, 54, 57, 55, 58, for example from SEQID NO: 1, 5, 8, 12, 15, 16, 42, 46 or 51 will in particular be presentessentially in one or more of the framework regions, FR1-L, FR2-L,FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4-H.

However, amino acid substitutions in one or more CDRs are also possible.

The invention also provides antibodies as defined above furthercomprising at least the variable domain of heavy chain and/or thevariable domain of light chain of one of the so-called anti-LAMP1antibodies MAb4.

Thus the invention relates in particular to an antibody which comprisesa variable domain of heavy chain of sequence SEQ ID NO: 88 or a sequenceat least 85% identical thereto and/or a variable domain of light chainof sequence of sequence SEQ ID NO: 89, or a sequence at least 85%identical thereto.

The antibody according to the invention is in particular a conventionalantibody, in particular a conventional monoclonal antibody, or anantibody fragment, a bispecific or multispecific antibody.

According to an embodiment, the antibody according to the inventioncomprises or consists of an IgG, or a fragment thereof.

The antibody of the invention and a fragment thereof may be,respectively, a murine antibody and a fragment of a murine antibody.

The antibody may also be a chimeric antibody, and in particular amurine/human antibody, e.g. an antibody comprising murine variabledomains of heavy and light chains and a CH domain and a CL domain from ahuman antibody. The antibody may be a fragment of such an antibody.

According to an embodiment, the antibody of the invention is:

-   -   a) a chimeric antibody comprising, or consisting of, a heavy        chain of sequence SEQ ID NO: 17 and/or a light chain of sequence        of sequence SEQ ID NO: 18 (i.e heavy and/or light chain of        chMAb1 as described in example 7); or    -   b) a chimeric antibody comprising, or consisting of, a heavy        chain of sequence SEQ ID NO: 19 and/or a light chain of sequence        of sequence SEQ ID NO: 20; (i.e heavy and/or light chain of        chMAb2 as described in example 7); or    -   c) a chimeric antibody comprising, or consisting of, a heavy        chain of sequence SEQ ID NO: 21 and/or a light chain of sequence        of sequence SEQ ID NO: 22; (i.e heavy and/or light chain of        chMAb2_(Can) as described in example 7); or    -   d) a chimeric antibody comprising, or consisting of, a heavy        chain of sequence SEQ ID NO: 49 and/or a light chain of sequence        of sequence SEQ ID NO: 50; (i.e heavy and/or light chain of        chMAb3); or    -   e) a chimeric antibody comprising, or consisting of, a heavy        chain of sequence SEQ ID NO: 49 and/or a light chain of sequence        of sequence SEQ ID NO:81; (i.e heavy and/or light chain of        chMAb3_VLR24-R93; or    -   f) a fragment of the chimeric antibody defined in a), b), c), d)        and e).

The antibody of the invention may also be a humanized antibody. Thus,according to an embodiment, the antibody of the invention comprises, orconsists of:

-   -   i) a heavy chain of sequence SEQ ID NO: 60 or a sequence at        least 85% identical thereto and/or a light chain of sequence of        sequence SEQ ID NO: 59 or a sequence at least 85% identical        thereto (i.e heavy and/or light chain of huMAb1_1 as described        in example 7.2); or    -   ii) a heavy chain of sequence SEQ ID NO: 62 or a sequence at        least 85% identical thereto and/or a light chain of sequence of        sequence SEQ ID NO: 61 or a sequence at least 85% identical        thereto (i.e heavy and/or light chain of huMAb1_2 as described        in example 7.2); or    -   iii) a heavy chain of sequence SEQ ID NO: 64 a or a sequence at        least 85% identical thereto nd/or a light chain of sequence of        sequence SEQ ID NO: 63 or a sequence at least 85% identical        thereto (i.e heavy and/or light chain of huMAb1_3 as described        in example 7.2).

In one embodiment the antibody of the invention is a humanized antibody.In a further embodiment said humanized antibody is obtained through theresurfacing technology. Such antibodies may also be called “resurfaced”antibodies.

The antibody according to the invention may also be a single domainantibody or a fragment thereof. In particular, a single domain antibodyfragment may consist of a variable heavy chain (VHH) which comprises theCDR1-H, CDR2-H and CDR3-H of the antibodies as described above. Theantibody may also be a heavy chain antibody, i.e. an antibody devoid oflight chain, which may or may not contain a CH1 domain.

The single domain antibody or a fragment thereof may also comprise theframework regions of a camelid single domain antibody, and optionallythe constant domain of a camelid single domain antibody.

The antibody according to the invention may also be an antibodyfragment, in particular a humanised antibody fragment, selected from thegroup consisting of Fv, Fab, F(ab′)2, Fab′, dsFv, (dsFv)2, scFv,sc(Fv)2, and diabodies.

The antibody may also be a bispecific or multispecific antibody formedfrom antibody fragments, at least one antibody fragment being anantibody fragment according to the invention. Multispecific antibodiesare polyvalent protein complexes as described for instance in EP 2 050764 A1 or US 2005/0003403 A1.

The bispecific or multispecific antibodies according to the inventioncan have specificity for (a) the first to third loops, in particular tothe first lumenal domain on human/Macaca fascicularis LAMP1 targeted byone of the so-called MAb1, MAb2, MAb2_(Can), MAb3, MAb3_VLR24-R93antibodies and (b) at least one other antigen. According to anembodiment the at least one other antigen is not a human or Macacafascicularis LAMP1 family member, and in particular not at least one orall of human and Macaca fascicularis LAMP2. According to anotherembodiment, the at least one other antigen may be an epitope on humanMacaca fascicularis LAMP1 other than said first to third loops, inparticular first lumenal domain targeted by one of the so-called MAb1,MAb2, MAb2_(Can) and MAb3 antibodies.

Said antibodies can be produced by any technique well known in the art.In particular said antibodies are produced by techniques as hereinafterdescribed.

Antibodies and fragments thereof according to the invention can be usedin an isolated (e.g., purified) from or contained in a vector, such as amembrane or lipid vesicle (e.g. a liposome).

The antibodies of the invention may represent any combination of theabove mentioned features.

Nucleic Acids, Vectors and Recombinant Host Cells

A further object of the invention relates to a nucleic acid sequencecomprising or consisting of a sequence encoding an antibody of theinvention as defined above.

Typically, said nucleic acid is a DNA or RNA molecule, which may beincluded in any suitable vector, such as a plasmid, cosmid, episome,artificial chromosome, phage or a viral vector.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence.

So, a further object of the invention relates to a vector comprising anucleic acid of the invention.

Such vectors may comprise regulatory elements, such as a promoter,enhancer, terminator and the like, to cause or direct expression of saidpolypeptide upon administration to a subject. Examples of promoters andenhancers used in the expression vector for animal cell include enhancerand promoter of human cytomegalovirus (Nelson, J., 1996 J. Virology 70:3207-3986), early promoter and enhancer of SV40 (Mizukami, T. and Itoh,S. et al., 1987, J Biochem. 101(5): 1307-1310), LTR promoter andenhancer of Moloney mouse leukemia virus (Kuwana Y. et al., 1987,Biochem Biophys Res Commun. 149: 960-968), promoter (Mason, J. O. etal., 1985, Cell 41: 479-487) and enhancer (Gillies, S. D. et al., 1983,Cell 33: 717-728) of immunoglobulin H chain and the like.

Any expression vector for animal cell can be used, so long as a geneencoding the human antibody C region can be inserted and expressed.Examples of suitable vectors include pAGE107 (Miyaji, H. et al., 1990,Cytotechnology 3(2): 133-140), pAGE103 (Mizukami, T. and Itoh, S. etal., 1987, J Biochem. 101(5): 1307-1310), pHSG274 (Brady, G. et al.,1984, Gene 27(2): 223-232), pKCR (O'Hare, K. et al., 1981, Proc NatlAcad Sci USA. 78(3): 1527-1531), pSG1 beta d2-4-(Miyaji, H. et al.,1990, Cytotechnology 4: 173-180) and the like.

Other examples of plasmids include replicating plasmids comprising anorigin of replication pCEP5, or integrative plasmids, such as forinstance pUC, pcDNA, pBR, and the like.

Other examples of viral vector include adenoviral, retroviral, herpesvirus and AAV vectors. Such recombinant viruses may be produced bytechniques known in the art, such as by transfecting packaging cells orby transient transfection with helper plasmids or viruses. Typicalexamples of virus packaging cells include PA317 cells, PsiCRIP cells,GPenv+cells, 293 cells, etc. Detailed protocols for producing suchreplication-defective recombinant viruses may be found for instance inWO 95/14785, WO 96/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No.6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94/19478.

A further object of the present invention relates to a cell which hasbeen transfected, infected or transformed by a nucleic acid and/or avector according to the invention.

The term “transformation” means the introduction of a “foreign” (i.e.extrinsic) gene, DNA or RNA sequence to a host cell, so that the hostcell will express the introduced gene or sequence to produce a desiredsubstance, typically a protein or enzyme coded by the introduced gene orsequence. A host cell that receives and expresses introduced DNA or RNAhas been “transformed”.

The nucleic acids of the invention may be used to produce a recombinantantibody of the invention in a suitable expression system. The term“expression system” means a host cell and compatible vector undersuitable conditions, e.g. for the expression of a protein coded for byforeign DNA carried by the vector and introduced to the host cell.

Common expression systems include E. coli host cells and plasmidvectors, insect host cells and Baculovirus vectors, and mammalian hostcells and vectors. Other examples of host cells include, withoutlimitation, prokaryotic cells (such as bacteria) and eukaryotic cells(such as yeast cells, mammalian cells, insect cells, plant cells, etc.).Specific examples include E. coli, Kluyveromyces or Saccharomycesyeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 313 cells,COS cells, HEK293 cells etc.) as well as primary or establishedmammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts,embryonic cells, epithelial cells, nervous cells, adipocytes, etc.).Examples also include mouse SP2/0-Ag14 cell (ATCC CRL1581), mouseP3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolatereductase gene (hereinafter referred to as “DHFR gene”) is defective(Urlaub, G. et al.; 1980, Proc Natl Acad Sci USA. 77(7): 4216-4220), ratYB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as“YB2/0 cell”), and the like. The YB2/0 cell is of interest, since ADCCactivity of chimeric or humanised antibodies is enhanced when expressedin this cell.

In particular, for expression of humanised antibody, the expressionvector may be either of a type in which a gene encoding an antibodyheavy chain and a gene encoding an antibody light chain exists onseparate vectors or of a type in which both genes exist on the samevector (tandem type). In respect of easiness of construction of ahumanised antibody expression vector, easiness of introduction intoanimal cells, and balance between the expression levels of antibody Hand L chains in animal cells, humanised antibody expression vector ofthe tandem type is preferred (Shitara, K. et al., 1994, J ImmunolMethods. January 3: 167(1-2): 271-8). Examples of tandem type humanisedantibody expression vector include pKANTEX93 (WO 97/10354), pEE18 andthe like.

The present invention also relates to a method of producing arecombinant host cell expressing an antibody according to the invention,said method comprising the steps consisting of: (i) introducing in vitroor ex vivo a recombinant nucleic acid or a vector as described aboveinto a competent host cell, (ii) culturing in vitro or ex vivo therecombinant host cell obtained and (iii), optionally, selecting thecells which express and/or secrete said antibody.

Such recombinant host cells can be used for the production of antibodiesof the invention.

Methods of Producing Antibodies of the Invention

Antibodies of the invention may be produced by any technique known inthe art, such as, without limitation, any chemical, biological, geneticor enzymatic technique, either alone or in combination.

Knowing the amino acid sequence of the desired sequence, one skilled inthe art can readily produce said antibodies or immunoglobulin chains, bystandard techniques for production of polypeptides. For instance, theycan be synthesized using well-known solid phase method, in particularusing a commercially available peptide synthesis apparatus (such as thatmade by Applied Biosystems, Foster City, Calif.) and following themanufacturer's instructions. Alternatively, antibodies andimmunoglobulin chains of the invention can be synthesized by recombinantDNA techniques as is well-known in the art. For example, these fragmentscan be obtained as DNA expression products after incorporation of DNAsequences encoding the desired (poly)peptide into expression vectors andintroduction of such vectors into suitable eukaryotic or prokaryotichosts that will express the desired polypeptide, from which they can belater isolated using well-known techniques.

In particular, the invention further relates to a method of producing anantibody of the invention, which method comprises the steps consistingof: (i) culturing a transformed host cell according to the invention;(ii) expressing said antibody or polypeptide; and (iii) recovering theexpressed antibody or polypeptide.

Methods for producing humanised or chimeric antibodies involveconventional recombinant DNA and gene transfection techniques are wellknown in the art (See Morrison, S. L. and Oi, V. T., 1984, Annu RevImmunol 2: 239-256 and patent documents U.S. Pat. No. 5,202,238; andU.S. Pat. No. 5,204,244).

In a particular embodiment, a chimeric antibody of the present inventioncan be produced by obtaining nucleic sequences encoding the murine VLand VH domains as previously described, constructing a chimeric antibodyexpression vector by inserting them into an expression vector for animalcell having genes encoding human antibody CH and human antibody CL, andexpressing the coding sequence by introducing the expression vector intoan animal cell.

In another particular embodiment, a humanised antibody of the presentinvention can be produced by obtaining nucleic sequences encodinghumanised VL and VH domains as previously described, constructing ahumanised antibody expression vector by inserting them into anexpression vector for animal cell having genes encoding human antibodyCH and human antibody CL, and expressing the coding sequence byintroducing the expression vector into an animal cell.

As the CH domain of a humanized or chimeric antibody, it may be anyregion which belongs to human immunoglobulin heavy chains, but those ofIgG class are suitable and any one of subclasses belonging to IgG class,such as IgG1, IgG2, IgG3 and IgG4, can also be used. Also, as the CL ofa human chimeric antibody, it may be any region which belongs to humanimmunoglobulin light chains, and those of kappa class or lambda classcan be used.

Antibodies can be humanised using a variety of techniques known in theart including, for example, the technique disclosed in the applicationWO2009/032661, CDR-grafting (EP 239,400; PCT publication WO91/09967;U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering orresurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka, G. M.et al., 1994, Protein Eng. 7(6): 805-814; Roguska, M. A. et al., 1994,Proc Natl Acad Sci USA 91(3): 969-973), and chain shuffling (U.S. Pat.No. 5,565,332) The general recombinant DNA technology for preparation ofsuch antibodies is also known (see European Patent Application EP 125023and International Patent Application WO 96/02576).

Antibodies of the invention are suitably separated from the culturemedium by conventional immunoglobulin purification procedures such as,for example protein A affinity chromatography, ceramic hydroxyapatitechromatography, mixed-mode chromatography, size-exclusion chromatographyetc.

The Fab of the present invention can be obtained by treating an antibodywhich specifically reacts with LAMP1 with a protease, such as papaine.Also, the Fab can be produced by inserting DNA sequences encoding bothchains of the Fab of the antibody into a vector for prokaryoticexpression, or for eukaryotic expression, and introducing the vectorinto procaryotic or eukaryotic cells (as appropriate) to express theFab.

The F(ab′)2 of the present invention can be obtained treating anantibody which specifically reacts with LAMP1 with a protease, pepsin.Also, the F(ab′)2 can be produced by binding Fab′ described below via athioether bond or a disulfide bond.

The Fab′ of the present invention can be obtained treating F(ab′)2 whichspecifically reacts with LAMP1 with a reducing agent, such asdithiothreitol. Also, the Fab′ can be produced by inserting DNAsequences encoding Fab′ chains of the antibody into a vector forprokaryotic expression, or a vector for eukaryotic expression, andintroducing the vector into prokaryotic or eukaryotic cells (asappropriate) to perform its expression.

The scFv of the present invention can be produced by taking sequences ofthe CDRs or VH and VL domains as previously described, constructing aDNA encoding an scFv fragment, inserting the DNA into a prokaryotic oreukaryotic expression vector, and then introducing the expression vectorinto prokaryotic or eukaryotic cells (as appropriate) to express thescFv. To generate a humanised scFv fragment, a well known technologycalled CDR grafting may be used, which involves selecting thecomplementary determining regions (CDRs) according to the invention, andgrafting them onto a human scFv fragment framework of known threedimensional structure (see, e. g., WO98/45322; WO 87/02671; U.S. Pat.No. 5,859,205; U.S. Pat. No. 5,585,089; U.S. Pat. No. 4,816,567;EP0173494).

The single chain antibody or VHH directed against LAMP1 may be obtainedfor instance by a method comprising the steps of (a) immunizing a mammalbelonging to the Camelidae with LAMP1 or a fragment thereof, so as toelicit antibodies (and in particular heavy chain antibodies) againstLAMP1; (b) obtaining a biological sample from the Camelidae thusimmunized, said sample comprising heavy chain antibody sequences and/orV_(HH) sequences that are directed against LAMP1; and (c) recovering(e.g isolating) heavy chain antibody sequences and/or VH_(H) sequencesthat are directed against LAMP1 from said biological sample. Suitablesingle chain antibody or VHH may also be obtained by screening a librarycomprising heavy chain antibody sequences and/or VHH sequences for heavychain antibody sequences and/or VHH sequences that compete for bindingto the first to third loops, in particular to the first lumenal domainof human and Macaca fascicularis LAMP1 proteins with an antibodycomprising the variable heavy and light chains of an antibody selectedfrom the group consisting of the so-called antibodies MAb1, MAb2,MAb2_(Can), MAb3, huMAb1_1 and huMAb1_2, huMAb1_3, for instance MAb1,MAb2, MAb2_(Can), MAb3.

Modification of the Antibodies of the Invention

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.

Modifications and changes may be made in the structure of the antibodiesof the present invention, and in the DNA sequences encoding them, andstill result in a functional antibody or polypeptide with desirablecharacteristics.

In making the changes in the amino sequences of polypeptide, thehydropathic index of amino acids may be considered. The importance ofthe hydropathic amino acid index in conferring interactive biologicfunction on a protein is generally understood in the art. It is acceptedthat the relative hydropathic character of the amino acid contributes tothe secondary structure of the resultant protein, which in turn definesthe interaction of the protein with other molecules, for example,enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophane (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate −3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

A further object of the present invention also encompassesfunction-conservative variants of the polypeptides of the presentinvention.

For example, certain amino acids may be substituted by other amino acidsin a protein structure without appreciable loss of activity. Since theinteractive capacity and nature of a protein define its biologicalfunctional activity, certain amino acid substitutions can be made in aprotein sequence, and of course in its DNA encoding sequence, whilenevertheless obtaining a protein with like properties. It is thuscontemplated that various changes may be made in the antibodiessequences of the invention, or corresponding DNA sequences which encodesaid polypeptides, without appreciable loss of their biologicalactivity.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. It is also possible to usewell-established technologies, such as alanine-scanning approaches, toidentify, in an antibody or polypeptide of the invention, all the aminoacids that can be substituted without significant loss of binding to theantigen. Such residues can be qualified as neutral, since they are notinvolved in antigen binding or in maintaining the structure of theantibody. One or more of these neutral positions can be substituted byalanine or by another amino acid can without changing the maincharacteristics of the antibody or polypeptide of the invention.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take several of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

It may be also desirable to modify the antibody of the invention withrespect to effector function, e.g. so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing inter-chain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and/or antibody-dependent cellular cytotoxicity (ADCC) (Caron,P. C. et al., 1992, J Exp Med. 176(4): 1191-1195 and Shopes B., 1992, JImmunol. 148(9): 2918-2922).

Another type of amino acid modification of the antibody of the inventionmay be useful for altering the original glycosylation pattern of theantibody, i.e. by deleting one or more carbohydrate moieties found inthe antibody, and/or adding one or more glycosylation sites that are notpresent in the antibody. The presence of either of the tripeptidesequences asparagine-X-serine, and asparagine-X-threonine, where X isany amino acid except proline, creates a potential glycosylation site.Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites).

Another type of modification of the antibody of the invention may be toremove a lysine in a CDR or specially close to a CDR since covalentattachment to cytotoxic via lysine side chain residue may interfere withbinding to antigen in the case of ADC.

Another type of modification involves the removal of sequencesidentified, either in silico or experimentally, as potentially resultingin degradation products or heterogeneity of antibody preparations. Asexamples, deamidation of asparagine and glutamine residues can occurdepending on factors such as pH and surface exposure. Asparagineresidues are particularly susceptible to deamidation, primarily whenpresent in the sequence Asn-Gly, and to a lesser extent in otherdipeptide sequences such as Asn-Ala. When such a deamidation site, inparticular Asn-Gly, is present in an antibody or polypeptide of theinvention, it may therefore be desirable to remove the site, typicallyby conservative substitution to remove one of the implicated residues.Such substitutions in a sequence to remove one or more of the implicatedresidues are also intended to be encompassed by the present invention.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, or tyrosine, (f) theamide group of glutamine. For example, such methods are described inWO87/05330.

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Sojahr, H. etal. (1987, Arch Biochem Biophys. 259(1): 52-57) and by Edge, A. S. etal. (1981, Anal Biochem. 118(1): 131-137). Enzymatic cleavage ofcarbohydrate moieties on antibodies can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura, N R. etal. (1987, Methods Enzymol 138: 350-359).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of non proteinaceous polymers, eg.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

Pharmaceutical Compositions

The antibodies or immunoconjugates of the invention may be combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form therapeuticcompositions.

Thus, another object of the invention relates to a pharmaceuticalcomposition comprising an antibody or an immunoconjugate of theinvention and a pharmaceutically acceptable carrier.

The invention also relates to a polypeptide or an immunoconjugateaccording to the invention, for use as a medicament.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and gender of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for atopical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous or intraocular administration and the like.

In particular, the pharmaceutical compositions contain vehicles whichare pharmaceutically acceptable for a formulation capable of beinginjected. These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The doses used for the administration can be adapted as a function ofvarious parameters, and in particular as a function of the mode ofadministration used, of the relevant pathology, or alternatively of thedesired duration of treatment.

To prepare pharmaceutical compositions, an effective amount of theantibody or immunoconjugate of the invention may be dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants,stabilizing agents, cryoprotectants or antioxidants. The prevention ofthe action of microorganisms can be brought about by antibacterial andantifungal agents. In many cases, it will be preferable to includeisotonic agents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with severalof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

The antibody or immunoconjugate of the invention of the invention may beformulated within a therapeutic mixture to comprise about 0.01 to 100milligrams, per dose or so.

Therapeutic Methods and Uses

The inventors have shown that an antibody directed against the first tothird loops of LAMP1, in particular against the first lumenal domain ofLAMP1, in particular MAb1 and MAb2 and Mab3, is able to activelyinternalize the LAMP1 receptor-antibody complex after binding andaccumulate probably via coated pits. Internalized antibodies MAb1, MAb2and Mab3 localized to early endosomes and subsequently trafficked to andaccumulation in lysosomal compartments.

ImageStream multispectral imaging flow cytometer (Amnis corp.) revealsthat the internalized antibodies accumulate in lysosomal compartments.Immunofluorescence analysis of viable Colo205 cells incubated with MAb1,MAb2 and MAb3 at 4° C. showed distinct plasma membrane staining.Incubation of cells at 37° C. with MAb1, MAb2 and MAb3 revealed labelingof both plasma membrane and intracellular vesicles after 4 hoursincubation. Since the internalization score (IS) revealing thefluorescence inside cells (as measured at 37° C.) is 10-fold higher thanthe fluorescence at the cell surface (as measured at 4° C.), this meansthat the LAMP1 protein is quickly recycling at cell membrane. Alltogether, our results show for the first time that LAMP1 might functionas a receptor mediating the internalization of antibodies and suggestthat availability of specific internalizing antibodies should aid indeveloping novel therapeutic methods to target toxins, drugs orshort-range isotopes to be delivered specifically to the interior of thecancer cells.

Furthermore, they have shown that an antibody according to theinvention, combined with a cytotoxic maytansinoid (DM4), inducescytotoxic activity in vitro on human HCT116 tumor or HEK293 cellscontaining a stable integration of the LAMP1 coding DNA sequence in thegenomic DNA wherein individual clones present different intensities ofLAMP1 on the cell surface.

In another example 9.4, the inventors showed that an antibody accordingto the invention, combined with a cytotoxic tomamycin dimer, inducescytotoxic activity in vitro.

They have also shown that an antibody combined with a cytotoxicmaytansinoid (DM4) induces a marked anti-tumor activity in vivo in amurine model of primary human colon adenocarcinoma xenografts derivedfrom patient, when used at a dose of 10 mg/kg, 5 mg/kg and 2.5 mg/kg,with a single injection at day 17 post tumor implantation as describedin example 10.1.1.

Furthermore, they have also shown that this immunoconjugate induces amarked anti-tumor activity in vivo in a murine model of primary humanlung tumor xenografts derived from patient, when used at a dose of 10mg/kg, 5 mg/kg and 2.5 mg/kg, with a single injection at day 26 posttumor implantation as described in example 10.1.2.

The inventors have also shown that the immunoconjugates ofDM4-SPDB-huMAb1_1, DM4-SPDB-chMAb2, DM4-SPDB-chMAb3 induce a markedanti-tumor activity in vivo in different murine model of differentcancer xenograft models as shown in example 10.2-10.4.

For example, it was shown the immunoconjugate DM4-SPDB-huMAb1_1 inducesa marked anti-tumor activity in vivo primary human invasive ductalcarcinoma xenograft and primary human lung tumor xenograft derived frompatient, when used at a dose of 10 mg/kg, 5 mg/kg and 2.5 mg/kg, with asingle injection, as described in example 10.2.2 and 10.2.3.

Also the immunoconjugates DM4-SPDB-chMAb2 and DM4-SPDB-chMAb3 induce amarked anti-tumor activity in primary human invasive ductal carcinomaxenograft derived from patient, when used at a dose of 10 mg/kg, 5 mg/kgand 2.5 mg/kg or 5 mg/kg, 2.5 mg/kg and 1.25 mg/kg, respectively, with asingle injection, as described in example 10.3.2 and 10.4.

Thus, polypeptides, antibodies, immunoconjugates, or pharmaceuticalcompositions of the invention may be useful for treating cancer.

The invention further relates to an anti-LAMP1 therapeutic agent for usefor treating cancer in a patient harboring LAMP1 gene copy number gainin cancer cells.

In an embodiment, said patient harboring LAMP1 gene copy number gain incancer cells has been selected by the in vitro method of selectingpatients with cancer according to the invention. In particular, the usecomprises selecting said patient harboring LAMP1 gene copy number gainin cancer cells by a method of selecting patients with cancer accordingto the invention.

The invention also relates to a method of treating a patient with cancerwhich comprises

-   -   a) selecting a patient with cancer who is likely to respond to        anti-LAMP1 therapy by a in vitro method of selecting patients        with cancer according to the invention; and    -   b) administering anti-LAMP1 therapy to said selected patient.

The invention further relates to a method of selecting a patient withcancer for anti-LAMP1 therapy, comprising:

-   -   (a) determining, in a biological sample of a patient with cancer        which includes cancer cells, if said patient harbors a LAMP1        gene copy number gain; and    -   (b) administering to said patient anti-LAMP1 therapy, if said        patient harbors a LAMP1 gene copy number gain.

The invention also relates to a method of treating cancer in a patient,comprising:

-   -   (a) determining, in a biological sample of a patient with cancer        which includes cancer cells, if said patient harbors a LAMP1        gene copy number gain; and    -   (b) administering to said patient anti-LAMP1 therapy, if said        patient harbors a LAMP1 gene copy number gain.

The cancer to be treated with antibodies, immunoconjugates, orpharmaceutical compositions of the invention is a cancer expressingLAMP1 on the cell surface, in particular overexpressing LAMP1 on thecell surface as compared to normal (i.e. non tumoral) cells of the sametissular origin.

Expression of LAMP1 by cancer cells may be readily assayed for instanceby using an antibody according to the invention, as described in thefollowing section “Diagnostic uses”, and in particular by animmunohistochemical method for instance as described in Example 5.

In particular the cancer may be colon adenocarcinomas but alsogastrointestinal tumors (small intestine, rectum, parotid gland), vitalorgans tumors (lung, liver, pancreas, stomach and kidney), reproductiveorgan tumors (breast, ovary and prostate) as well as skin, larynx andsoft tissue tumors, for instance the cancer is selected from the groupconsisting of colon adenocarcinoma, gastrointestinal tumors (smallintestine, rectum, parotid gland), vital organs tumors (lung, liver,pancreas and kidney), reproductive organ tumors (breast, ovary andprostate) as well as skin, larynx or soft tissue tumors.

In one embodiment gastrointestinal tumors are small intestine tumor,rectum tumor and/or parotid gland tumor.

In one embodiment reproductive organ tumors gastrointestinal tumors arelung tumor, liver tumor, pancreas tumor, stomach tumor and kidney tumor.

In one embodiment reproductive organ tumors are breast tumor, ovarytumor or prostate tumor.

Screening of a panel of human tumors by immunohistochemistry using amouse anti-human LAMP1 antibody according to the invention indeed showedantibody staining in these types of cancers, as described in furtherdetails in Example 5.

In particular, LAMP1 expressing human tumoral cells may be selected fromthe group consisting of colon, stomach, rectum, lung squamous cellcarcinoma, breast invasive ductal and lobular carcinoma and prostateadenocarcinoma cells. These tumors were indeed found to display morethan 50% of cells positive for LAMP1 expression at the cell membrane(see example 5).

The antibodies or immunoconjugates of the invention may be used alone orin combination with any suitable growth-inhibitory agent.

The antibodies of the invention may be conjugated or linked to a growthinhibitory agent, cytotoxic agent, or a prodrug-activating enzyme aspreviously described. Antibodies of the invention may be indeed usefulfor targeting said growth inhibitory agent, cytotoxic agent, or aprodrug to the cancerous cells expressing or over-expressing LAMP1 ontheir surface.

It is also well known that therapeutic monoclonal antibodies can lead tothe depletion of cells bearing the antigen specifically recognized bythe antibody. This depletion can be mediated through at least threemechanisms: antibody mediated cellular cytotoxicity, complementdependent lysis, and direct anti-tumour inhibition of tumour growththrough signals given via the antigen targeted by the antibody.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system to antibodies which are bound to their cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al. (1997) may be performed.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted antibodies bound onto Fcreceptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer(NK) cells, neutrophils, and macrophages) enable these cytotoxiceffector cells to bind specifically to an antigen-bearing target celland subsequently kill the target cell. To assess ADCC activity of amolecule of interest, an in vitro ADCC assay, such as that described inU.S. Pat. No. 5,500,362 or 5,821,337 was also contemplated. It is knownto the skilled in the art that specific mutations such as the D265Amutation according to the nomenclature described by Kabat et al.(Sequences of Proteins of Immunological Interest, 5th edition, NationalInstitute of Health, Bethesda, Md., 1991) significantly decrease bindingto Fc□Rs and ADCC (Lund et al., J. Immunol., 157:4963-4969, 1996;Shields et al., J. Biol. Chem., 276(1): 6591-6604, 2001).

In one example the mutation of 266A of for example SEQ ID NO: 56 in thehulgG1 corresponds to the D265A mutation mentioned above and thussignificantly decrease binding to Fc□Rs and ADCC.

Thus, an object of the invention relates to a method for treating acancer comprising administering a subject in need thereof with atherapeutically effective amount of a polypeptide, an antibody, animmunoconjugate or a pharmaceutical composition of the invention.

“Antibody-dependent cellular phagocytosis” or “ADCP” refers to a form ofcytotoxicity in which antibodies bound onto Fc receptors (FcRs) presenton certain cytotoxic cells (e.g. macrophages) enable these cytotoxiceffector cells to bind specifically to an antigen-bearing target celland subsequently kill the target cell by phagocytosis. To assess ADCPactivity of a molecule of interest, an in vitro ADCP assay, such as thatdescribed in McEarchem et al., 2007, Blood 109:1185.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, inhibiting the progress of,or preventing the disorder or condition to which such term applies, orone or more symptoms of such disorder or condition. By the term“treating cancer” as used herein is meant the inhibition of the growthof malignant cells of a tumour and/or the progression of metastases fromsaid tumor. Such treatment can also lead to the regression of tumorgrowth, i.e., the decrease in size of a measurable tumor. In particular,such treatment leads to the complete regression of the tumor ormetastase.

According to the invention, the term “patient” or “patient in needthereof” is intended for a human or non-human mammal affected or likelyto be affected with a malignant tumor. In particular, said patient maybe a patient who has been determined to be susceptible to a therapeuticagent targeting LAMP1, in particular to an antibody or immunoconjugateaccording to the invention, for instance according to a method asdescribed herebelow.

As disclosed above, “anti-LAMP1 therapy” is a therapy which involves atherapeutic agent targeting LAMP1. According to the invention, the term“therapeutic agent targeting LAMP1” or “anti-LAMP1 therapeutic agent”describe an agent binding to LAMP1 and having cytotoxic and/orcytostatic activity.

As used herein, the term “binding agent” refers to a molecule thatexhibits specific binding activity towards LAMP1. Such a bindingmolecule can include a variety of different types of moleculesincluding, for example, macromolecules and small organic molecules.Small molecule binding agents can include, for example, receptorligands, antagonists and agonists. Macromolecules can include, forexample, peptide, polypeptide and protein, nucleic acids encodingpolypeptide binding agents, lectins, carbohydrate and lipids. It isunderstood that the term includes fragments and domains of the agent solong as binding function is retained. Similarly, the boundaries of thedomains are not critical so long as binding activity is maintained. Inthe specific example where the binding agent is a peptide, polypeptideor protein, such binding proteins can include monomeric or multimericspecies. Heteromeric binding proteins are a specific example ofmultimeric binding proteins. It is understood that when referring tomultimeric binding proteins that the term includes fragments of thesubunits so long as assembly of the polypeptides and binding function ofthe assembled complex is retained. Heteromeric binding proteins include,for example, antibodies and fragments thereof such as Fab and F(ab′)2portions.

According to an embodiment, the anti-LAMP1 therapeutic agent is ananti-LAMP1 antibody or an immunoconjugate comprising an anti-LAMP1antibody and at least one growth inhibitory agent.

By a “therapeutically effective amount” of the polypeptide of theinvention is meant a sufficient amount of the polypeptide to treat saidcancer disease, at a reasonable benefit/risk ratio applicable to anymedical treatment. It will be understood, however, that the total dailyusage of the polypeptides and compositions of the present invention willbe decided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically effective dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; activity of thespecific polypeptide employed; the specific composition employed, theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific polypeptide employed; the duration of the treatment; drugs usedin combination or coincidental with the specific polypeptide employed;and like factors well known in the medical arts. For example, it is wellknown within the skill of the art to start doses of the compound atlevels lower than those required to achieve the desired therapeuticeffect and to gradually increase the dosage until the desired effect isachieved. Another object of the invention relates to a polypeptide, anantibody, an immunoconjugate or a pharmaceutical composition of theinvention for use in the treatment of a malignant tumour.

In particular, the polypeptide, antibody, immunoconjugate orpharmaceutical composition may be used for inhibiting the progression ofmetastases of a malignant tumour.

Polypeptides of the invention may be used in combination with any othertherapeutical strategy for treating malignant tumour (e.g. adjuvanttherapy), and/or for reducing the growth of the metastatic tumour.

Efficacy of the treatment with an antibody or immunoconjugate accordingto the invention may be readily assayed in vivo, for instance on a mousemodel of cancer and by measuring e.g. changes in tumor volume betweentreated and control groups, % tumor regression, partial regressionand/or complete regression as defined in Example 10.

In one embodiment, the antibody is one of the anti-LAMP1 antibodiesdeveloped by the applicant (the so-called antibodies “MAb1”, “MAb2”,“MAb3”, huMAb1_1 and huMAb1_2, huMAb1_3) that bind specifically to humanLAMP1 and distinguish tumoral from non-tumoral tissues as furtherdescribed in the section “Antibodies” above.

Diagnostic Uses

The antibody according to the invention revealed that some LAMP1expression occurred at the membrane of non-tumoral cells but wasrestricted to stomach epithelial cells, oesophageal epithelial cells,breast epithelial cells, prostate epithelial cells, testicularepithelial cells and limited to a few cells. Nevertheless, prevalenceand mean intensities for LAMP1 expression at the membrane of non-tumoralsamples were lower than those found in tumours.

Instead, LAMP1 is highly expressed at the surface of a variety othercarcinomas than colon adenocarcinomas, including gastrointestinal tumors(small intestine, rectum, parotid gland), vital organs tumors (lung,liver, stomach, pancreas and kidney), reproductive organ tumors (breast,ovary and prostate) as well as skin, larynx and soft tissue tumors, forexample gastrointestinal tumors (small intestine, rectum, parotidgland), vital organs tumors (lung, liver, pancreas and kidney),reproductive organ tumors (breast, ovary and prostate) as well as skin,larynx and soft tissue tumors. Therefore, LAMP1 constitutes a marker ofcertain cancers and, therefore, has the potential to be used to indicatethe effectiveness of an anti-cancer therapy or detecting recurrence ofthe disease.

In particular, LAMP1 is highly expressed at the surface of carcinomasselected from the group consisting of colon, rectum, lung squamous cellcarcinoma, stomach, breast invasive ductal and lobular carcinoma andprostate adenocarcinoma cells, more particularly colon, rectum, lungsquamous cell carcinoma, breast invasive ductal and lobular carcinomaand prostate adenocarcinoma cells.

As described above in the chapter ‘antibodies’, the inventors developedantibodies MAb1, MAb2, MAb3 allowing for the first time to detectextracellularly expressed LAMP1 and thus to perform IHC analysis onFrozen-OCT (from Optimal Cutting Temperature) specimens and AFA (AlcoholFormalin Acetic acid Fixative) and MAb4 allowing LAMP1 reinforcement inFFPE format and thus allows to distinguish cancerous from non-canceroustissue.

In a preferred embodiment, the antibody of the invention is used ascomponent of an assay in the context of a therapy targeting LAMP1expressing tumours, in order to determine susceptibility of the patientto the therapeutic agent, monitor the effectiveness of the anti-cancertherapy or detect recurrence of the disease after treatment. Inparticular, the same antibody of the invention is used both as componentof the therapeutic agent and as component of the diagnostic assay.

Thus, a further object of the invention relates to an antibody accordingto the invention for use for in vivo detecting LAMP1 expression in asubject, or for use for ex vivo detecting LAMP1 expression in biologicalsample of a subject. Said detection may be intended in particular for

-   -   a) diagnosing the presence of a cancer in a subject, or    -   b) determining susceptibility of a patient having cancer to a        therapeutic agent targeting LAMP1, in particular an        immunoconjugate according to the invention, or    -   c) monitoring effectiveness of anti-LAMP1 cancer therapy or        detecting cancer relapse after anti-LAMP1 cancer therapy, in        particular for therapy with an immunoconjugate according to the        invention by detecting expression of the surface protein LAMP1        on tumor cells.

In an embodiment, the antibody is intended for an in vitro or ex vivouse. For example, LAMP1 may be detected in vitro or ex vivo in abiological sample obtained from a subject, using an antibody of theinvention. The use according to the invention may also be an in vivouse. For example, an antibody according to the invention is administeredto the subject and antibody-cell complexes are detected and/orquantified, whereby the detection of said complexes is indicative of acancer.

The invention further relates to an in vitro or ex vivo method ofdetecting the presence of a cancer in a subject, comprising the stepsconsisting of:

-   -   a) contacting a biological sample of a subject with an antibody        according to the invention, in particular in conditions        sufficient for the antibody to form complexes with said        biological sample,    -   b) measuring the level of antibody bound to said biological        sample,    -   c) detecting the presence of a cancer by comparing the measured        level of bound antibody with a control, an increased level of        bound antibody compared to control being indicative of a cancer.

The invention also relates to an in vitro or ex vivo method ofdetermining susceptibility of a patient having cancer to a therapeuticagent targeting LAMP1, in particular to an immunoconjugate according tothe invention, which method comprises the steps consisting of:

-   -   a) contacting a biological sample of a patient having cancer        with an antibody according to the invention, in particular in        conditions sufficient for the antibody to form complexes with        said biological sample,    -   b) measuring the level of antibody bound to said biological        sample,    -   c) comparing the measured level of bound antibody to said        biological sample sample with the level of antibody bound to a        control,    -   wherein an increased level of bound antibody to said biological        sample compared to control is indicative of a patient        susceptible to a therapeutic agent targeting LAMP1.

In the above methods, said control can be a normal, non cancerous,biological sample of the same type, or a reference value determined arepresentative of the antibody binding level in normal biological sampleof the same type. In an embodiment, the antibodies of the invention areuseful for diagnosing a LAMP1 expressing cancer, such as a colonadenocarcinoma, gastrointestinal tumors (small intestine, rectum,parotid gland), vital organs tumors (lung, liver, pancreas and kidney),reproductive organ tumors (breast, ovary and prostate) as well as skin,larynx and soft tissue tumors.

The invention further relates to an in vitro or ex vivo method ofmonitoring effectiveness of anti-LAMP1 cancer therapy, comprising thesteps consisting of:

-   -   a) contacting a biological sample of a subject undergoing        anti-LAMP1 cancer therapy, with an antibody according to the        invention, in particular in conditions sufficient for the        antibody to form complexes with said biological sample,    -   b) measuring the level of antibody bound to said biological        sample,    -   c) comparing the measured level of bound antibody with the level        of antibody bound to a control;    -   wherein a decreased level of bound antibody to said biological        sample compared to control is indicative of effectiveness of        said anti-LAMP1 cancer therapy.

In said method, an increased level of bound antibody to said biologicalsample compared to control is indicative of ineffectiveness of saidanti-LAMP1 cancer therapy.

Said control is in particular a biological sample of the same type asthe biological sample submitted to analysis, but which was obtained fromthe subject previously in time, during the course of the anti-LAMP1cancer therapy.

The invention further relates to an in vitro or ex vivo method ofdetecting cancer relapse after anti-LAMP1 cancer therapy, comprising thesteps consisting of:

-   -   (a) contacting a biological sample of a subject having completed        anti-LAMP1 cancer therapy, with an antibody according to the        invention, in particular in conditions sufficient for the        antibody to form complexes with said biological sample,    -   (b) measuring the level of antibody bound to said biological        sample,    -   (c) comparing the measured level of bound antibody with the        level of antibody bound to a control,    -   wherein a increased level of bound antibody to said biological        sample compared to control is indicative of cancer relapse after        anti-LAMP1 cancer therapy.

Said control is in particular a biological sample of the same type asthe biological sample submitted to analysis, but which was obtained fromthe subject previously in time, upon or after completion of theanti-LAMP1 cancer therapy.

Said anti-LAMP1 cancer therapy is in particular a therapy using anantibody or immunoconjugate according to the invention. Said anti-LAMP1cancer therapy targets a LAMP1 expressing cancer, in particular a colonadenocarcinoma, gastrointestinal tumors (small intestine, rectum,parotid gland), vital organs tumors (lung, liver, pancreas, stomach andkidney), reproductive organ tumors (breast, ovary and prostate) as wellas skin, larynx and soft tissue tumors.

In an embodiment, antibodies of the invention may be labelled with adetectable molecule or substance, such as a fluorescent molecule, aradioactive molecule or any other labels known in the art that provide(either directly or indirectly) a signal.

As used herein, the term “labeled”, with regard to the antibodyaccording to the invention, is intended to encompass direct labeling ofthe antibody by coupling (i.e., physically linking) a detectablesubstance, such as a radioactive agent or a fluorophore (e.g.fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine(Cy5)) to the polypeptide, as well as indirect labeling of thepolypeptide by reactivity with a detectable substance.

An antibody of the invention may be labelled with a radioactive moleculeby any method known to the art. For example radioactive moleculesinclude but are not limited radioactive atom for scintigraphic studiessuch as I¹²³, I¹²⁴, I¹²⁵, In¹¹¹, Re¹⁸⁶, Re¹⁸⁸, Tc⁹⁹, and isotopes forPositron Emission Tomography such as Zr⁸⁹, I¹²⁴, Ga⁶⁸ or Cu⁶⁴.

A “biological sample” encompasses a variety of sample types obtainedfrom a subject and can be used in a diagnostic or monitoring assay.Biological samples include but are not limited to blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom, and the progenythereof. Therefore, biological samples encompass clinical samples, cellsin culture, cell supernatants, cell lysates, serum, plasma, biologicalfluid, and tissue samples, in particular tumor sample.

In particular, the biological tissues may be prepared as frozen-OCT(Optimal Cutting Temperature) or AFA (acetic formalin alcohol) samples.Indeed, antibodies according to the invention can advantageously be usedon AFA sample which is a format used by hospitals to collect and archivetissue samples.

Measuring or determining the level of antibody bound the said biologicalsample may be performed by any suitable method known in the art such asFACS or IHC, for instance.

The invention also relates to an in vivo method of detecting thepresence of a cancer in a subject, comprising the steps consisting of:

-   -   a) administering an antibody according to the invention        detectably labelled to a patient,    -   b) detecting localisation of said detectably labelled antibody        in the patient by imaging.

Antibodies of the invention may be useful for staging of cancer (e.g.,in radioimaging). They may be used alone or in combination with othercancer markers.

The terms “detection” or “detected” as used herein includes qualitativeand/or quantitative detection (measuring levels) with or withoutreference to a control.

In the content of the invention, the term “diagnosing”, as used herein,means the determination of the nature of a medical condition intended toidentify a pathology which affects the subject from a number ofcollected data.

In said method, the cancer is a LAMP1 expressing cancer, in particular acolon adenocarcinoma, gastrointestinal tumors (small intestine, rectum,parotid gland), vital organs tumors (lung, liver, pancreas and kidney),reproductive organ tumors (breast, ovary and prostate) as well as skin,larynx and soft tissue tumors.

Method of Selecting Patients with Cancer

The invention relates to an in vitro method of selecting patients withcancer which comprises:

-   -   a) determining, in a biological sample of a patient with cancer        which includes cancer cells, if said patient harbors a LAMP1        gene copy number gain; and    -   b) selecting the patient based on the presence of LAMP1 gene        copy number gain.

In an embodiment, said method is for selecting a patient with cancer whois likely to respond to anti-LAMP1 therapy, and said patient is selectedas likely to respond to anti-LAMP1 therapy if said patient harbors aLAMP1 gene copy number gain. If said patient does not harbor a LAMP1gene copy number gain, the patients may nevertheless be selected aslikely to respond to anti-LAMP1 therapy based, for instance, on thedetection of cell surface expression of LAMP1 as expression oroverexpression of LAMP1 at the surface of tumor cells may have othercauses than LAMP1 gene copy number gain.

The LAMP1 gene gain can be related to a focal somatic gain oramplification, a somatic large region gain or amplification on 13 q, asomatic chromosome duplication, a somatic chromosome triplication orpolyploidy. LAMP1 gene copy number gain or amplification is included ina larger amplicon. The term “amplicon” as used herein refers to asegment of the genome that forms multiple linear copies. According tothe invention, the amplicon which might undergo copy number variationleading to a LAMP1 gene copy number gain will be called herein LAMP1amplicon.

Said “LAMP1 amplification” comprises a DNA region which can measurebetween 378 kb and 34.2 MB. Said “LAMP1 gain” comprises a DNA regionwhich can measure between 523 kb and 95.8 MB

In one embodiment, the LAMP1 gene copy number gain can be signified bythe CNV of a LAMP1 amplicon in colon PDX which comprises at least 454 kbfrom base 113785387 to base 114240314 on human chromosome 13(NC_000013). Said minimal LAMP1 amplicon contains others genes thanLAMP1, for example the genes ADPRHL1, CUL4A, DCUNID2, GRTP1, LAMP1,LOC100130463, PCID2, PRO7, TFDP1, TMCO3 and F10.

In another embodiment said LAMP1 amplicon comprises at least the genesADPRHL1, ATP11A, ATP4B, CUL4A, DCUN1D2, F10, F7, FAM70B, FLJ41484,FLJ44054, GAS6, GRK1, GRTP1, LAMP1, LINC00552, LOC100128430,LOC100130463, LOC100506063, LOC100506394, MCF2L, MCF2L-AS1, PCID2, PROZ,RASA3, TFDP1 and TMCO3C13orf35.

In a further embodiment the LAMP1 amplicon comprises 95.8 Mb from base19,296,544 to base 115,107,245 on human chromosome 13 (NC_000013).

In the context of the present invention, the positions of thenucleotides are indicated accordingly to the NCBI human genome sequence(Build 37, February 2009). It is known to the one skilled in the art,that a genome sequence is variable from an individual to another.Therefore, the positions defined herein for the LAMP1 amplicon mayslightly change according to the human genome sequence used. However,methods to compare genomic sequences and nucleotide positions are wellknown to the one skilled in the art.

There are numerous methods allowing determining the presence of a LAMP1gene copy number change in biological samples which are well known fromthe one skilled in the art. These methods include, without beinglimited, hybridization methods with DNA probes specific of markersequences, such as comparative genomic hybridization (CGH), matrix-CGH,array-CGH, oligonucleotide arrays, representational oligonucleotidemicroarray (ROMA), high-throughput technologies for SNP genotyping, forexample Affymetrix SNP chips, and amplification methods such asquantitative PCR.

In particular, the presence of said marker LAMP1 gene copy number changeis determined by amplification, or by hybridization with DNA probesspecific for LAMP1 gene or genes included in the LAMP1 amplicon. In anembodiment, the method of the invention is implemented by FluorescenceIn Situ Hybridization (FISH), Comparative Genomic Hybridization (CGH),New Generation Sequencing (NGS) and/or Polymerase Chain Reaction (PCR).

Accordingly, the invention relates to a method, wherein LAMP1 gene copynumber gain is determined with a method selected from the groupconsisting of FISH, CGH, NGS and/or PCR.

Methods of quantitative PCR are well-known in the art and includereal-time PCR, competitive PCR and radioactive PCR. For instance, aquantitative PCT method to enumerate DNA copy number has been describedin the U.S. Pat. No. 6,180,349.

As used herein, the term “primer” refers to an oligonucleotide which iscapable of annealing to a target sequence and serving as a point ofinitiation of DNA synthesis under conditions suitable for amplificationof the primer extension product which is complementary to said targetsequence. The primer is typically single stranded for maximum efficiencyin amplification. In particular, the primer is anoligodeoxyribonucleotide. The length of the primer depends on severalfactors, including temperature and sequence of the primer, but must belong enough to initiate the synthesis of amplification products. In anembodiment the primer is from 15 to 35 nucleotides in length. A primercan further contain additional features which allow for detection,immobilization, or manipulation of the amplified product. The primer mayfurthermore comprise covalently-bound fluorescent dyes, which conferspecific fluorescence properties to the hybrid consisting of the primerand the target-sequence or non covalently-bound fluorescent dyes whichcan interact with the double-stranded DNA/RNA to change the fluorescenceproperties. Fluorescent dyes which can be used are for exampleSYBR-green or ethidium bromide.

A “pair of primers” or “primer pair” as used herein refers to oneforward and one reverse primer as commonly used in the art of DNAamplification such as in PCR amplification.

As used herein, a “probe” refers to an oligonucleotide capable ofbinding in a base-specific manner to a complementary strand of nucleicacid. A probe may be labeled with a detectable moiety. Various labelingmoieties are known in the art. Said moiety may, for example, either be aradioactive compound, a detectable enzyme (e.g., horse radish peroxidase(HRP)) or any other moiety capable of generating a detectable signalsuch as calorimetric, fluorescent, chemiluminescent orelectrochemiluminescent signal. The detectable moiety may be detectedusing known methods. A probe may vary in length from about 5 to 100nucleotides, for instance from about 10 to 50 nucleotides, or from about20 to 40 nucleotides. In an embodiment, a probe comprises 33nucleotides.

The terms “hybridize” or “hybridization,” as is known to those skilledin the art, refer to the binding of a nucleic acid molecule to aparticular nucleotide sequence under suitable conditions, namely understringent conditions.

The term “stringent condition” or “high stringency condition” as usedherein corresponds to conditions that are suitable to produce bindingpairs between nucleic acids having a determined level ofcomplementarity, while being unsuitable to the formation of bindingpairs between nucleic acids displaying a complementarity inferior tosaid determined level. Stringent conditions are the combination of bothhybridization and wash conditions and are sequence dependent. Theseconditions may be modified according to methods known from those skilledin the art (Tijssen, 1993, Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, New York). Generally, highstringency conditions are selected to be about 5° C. lower than thethermal melting point (Tm), for instance at a temperature close to theTm of perfectly base-paired duplexes (Andersen, Nucleic acidHybridization, Springer, 1999, p. 54). Hybridization procedures are wellknown in the art and are described for example in Ausubel, F. M., Brent,R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl,K. eds. (1998) Current protocols in molecular biology. V. B. Chanda,series ed. New York: John Wiley & Sons.

High stringency conditions typically involve hybridizing at about 50° C.to about 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS, and washing in0.2×SSC/0.1% SDS at about 60° C. to about 68° C.

In one embodiment, the invention relates to a method wherein the meanLAMP1 gene copy number in cancer cells is ≥2.5. In particular the meanLAMP1 gene copy number in cancer cells may be 2.5 and <5.

In one embodiment, the invention relates to a method wherein the meanLAMP1 gene copy number in cancer cells is ≥5.

The method of the invention can further comprise determining if LAMP1 isexpressed at the surface of cancer cells of the patient, and i) saidpatient is selected as likely to respond to anti-LAMP1 therapy if saidpatient harbors a LAMP1 gene copy number gain and if said cancer cellsof the patient express LAMP1 at their surface or ii) said patient isselected as unlikely to respond to anti-LAMP1 therapy if said cancercells of the patient do not express LAMP1 at their surface.

There are numerous methods allowing determining if LAMP1 is expressed atthe surface of cancer cells, or overexpressed as compared with normalcells (i.e. non tumoral) of the same tissular origin, as which are wellknown from the one skilled in the art. These methods include forexample, without being limited, IHC, Western Blot (WB), Fluorescenceactivated cell sorting (FACS) analysis, immunofluorescence (IF),immunoprecipitation (IP) and Enzyme-linked immunosorbent assay (ELISA).

In an embodiment, immunohistochemistry (IHC) is used for determining ifLAMP1 is expressed or over-expressed at the surface of cancer cells.

Expression of LAMP1 by cancer cells may be readily assayed for instanceby using an anti-LAMP1 antibody as described in example 3.

The inventors showed that, LAMP1 gain is detected in 28 tumor types,including Colorectal adenocarcionoma, Stomach, Liver, Lung(Adenocarcinoma and Squamous), Breast (Basal, BRCA, LUMA, LUMB andHER2), Ovary, Head & neck, Kidney (Kidney Chromophobe, Kidney RenalClear Cell Carcinoma, Kidney Renal Papilliary), Cell Carcinoma, Cervicalsquamous Cell, Pancreatic, Prostate, Bladder urothelial, Glioma (Lowgrade glyoma and Glioblastoma multiform), Uterus, Thyroid, Leukemia,Lymphoma, Esophageal, Melanoma and Soft tissue sarcoma. High gain oramplification is detected in, breast, cervical, colorectal,glioblastoma, head and neck, liver, lung, glioma, ovarian, stomach anduterine cancer.

Accordingly, in an embodiment of the method of the invention, thepatient is having a cancer selected from the group consisting ofcolorectal, stomach, liver, lung, breast, ovarian, head and neck,kidney, pancreatic, prostate, uterine, glioma, bladder, thyroid cancerand leukemia, lymphoma, esophageal, melanoma and soft tissue sarcoma,for instance colorectal, stomach, liver, lung, ovarian, head and neck,kidney, pancreatic, prostate, uterine, glioma, bladder, thyroid cancerand leukemia, lymphoma, esophageal, melanoma and soft tissue sarcoma.

In a further embodiment the patient is having a cancer selected from thegroup consisting of, breast, cervical, colorectal, glioblastoma, headand neck, liver, lung, glioma, ovarian, stomach, and uterine cancer; orin particular from the group consisting of cervical, colorectal,glioblastoma, head and neck, liver, lung, glioma, ovarian, stomach,thyroid, and uterine cancer; or still more particularly from the groupconsisting of colon and lung cancer.

LAMP1 gene copy number gain and high expression of LAMP1 could bedetected at the surface of cancers selected from the group consisting ofcolon, lung, liver, pancreatic, kidney breast, ovarian, prostate,stomach cancer.

Thus in one embodiment the cancer may be selected from colon, lung,liver, pancreatic, kidney, ovarian, prostate, stomach cancer, forexample from colon, lung, liver, pancreatic, kidney, ovarian, prostate,stomach cancer.

Furthermore, LAMP1 gene copy gain is correlated with the LAMP1 mRNAexpression in bladder, breast, colon, lung, stomach and ovarian cancer.A significant association is shown between LAMP1 gene copy numbergain/amplification and the expression of LAMP1 at the plasma membrane oftumor cells for colon, stomach and lung tumor PDX.

Accordingly, in a further embodiment of the method of the invention, thepatient is having a cancer selected from the group consisting of breast,colon, lung, stomach, and ovarian. In another embodiment, the patient ishaving a cancer selected from the group consisting of colon, lung,stomach and ovarian.

An “anti-LAMP1 therapy” is a therapy which involves a therapeutic agenttargeting LAMP1. In one embodiment, such an anti-LAMP1 therapy is ananti-LAMP1 antibody or immunoconjugate. Anti-LAMP1 therapy is describedin further details hereafter.

The cancer may be in particular bladder, cervical, colorectal,glioblastoma, head and neck, kidney, liver, lung, glioma, ovarian,pancreatic, prostate, stomach, thyroid, and uterine cancer. In anotherexample the cancer is particularly colorectal or lung cancer.

Kits

Finally, the invention also provides kits comprising at least oneantibody or immunoconjugate of the invention. Kits containing antibodiesof the invention find use in detecting the surface protein LAMP1, or intherapeutic or diagnostic assays. Kits of the invention can contain apolypeptide or antibody coupled to a solid support, e.g. a tissueculture plate or beads (e.g. sepharose beads). Kits can be providedwhich contain antibodies for detection and quantification of the surfaceprotein LAMP1 in vitro, e.g. in an ELISA or a Western blot. Such anantibody useful for detection may be provided with a label such as afluorescent or radiolabel.

The invention will be further illustrated in light of the followingFigures and Examples.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the VH sequence of the “MAb1” antibody.

SEQ ID NO: 2-4 show the sequences of the CDR1-H, CDR2-H, CDR3-H of the“MAb1” antibody.

SEQ ID NO: 5 shows the VL sequence of the “MAb1” antibody.

SEQ ID NO: 6-7 show the sequences of the CDR1-L, CDR3-L of the “MAb1”antibody.

SEQ ID NO: 8 shows the VH sequence of the “MAb2” antibody.

SEQ ID NO: 9-11 show the sequences of the CDR1-H, CDR2-H, CDR3-H of the“MAb2” antibody.

SEQ ID NO: 12 shows the VL sequence of the “MAb2” antibody.

SEQ ID NO: 13-14 show the sequences of the CDR1-L, CDR3-L of the “MAb2”antibody.

SEQ ID NO: 15 shows the VH sequence of the so-called “Mab2_(Can)”antibody.

SEQ ID NO: 16 shows the VL sequence of the so-called “Mab2_(Can)”antibody.

SEQ ID NO: 17 shows the sequence of the heavy chain of the chimericantibody “chMAb1” antibody.

SEQ ID NO: 18 shows the sequence of the light chain of the chimericantibody “chMAb1” antibody.

SEQ ID NO: 19 shows the sequence of the heavy chain of the chimericantibody “chMAb2” antibody.

SEQ ID NO: 20 shows the sequence of the light chain of the chimericantibody “chMAb2” antibody.

SEQ ID NO: 21 shows the sequence of the heavy chain of the chimericantibody “chMab2_(Can)” antibody.

SEQ ID NO: 22 shows the sequence of the light chain of the chimericantibody “chMab2_(Can)” antibody.

SEQ ID NO: 23 shows the DNA sequence of full-length human LAMP1 asavailable from GenBank database under accession number NM 005561.3.

SEQ ID NO: 24 shows the Protein sequence of full-length human LAMP1 asavailable from GenBank database under NP_005552.3.

SEQ ID NO: 25 shows the Protein sequence of full-length mouse LAMP1 asavailable from GenBank database under NP_034814

SEQ ID NO: 26 shows the Protein sequence of full-length rat LAMP1 asavailable from GenBank database under NP_036989.

SEQ ID NO: 27 shows the Protein sequence of full-length Macaca mulattaLAMP1 as available from GenBank database under XP_002723509.

SEQ ID NO: 28 shows the sequence of human LAMP1 extracellular domainwithout Peptide Signal, followed by C-terminal 6 amino acid His-Tag.

SEQ ID NO: 29 shows the sequence of cynomologous monkey LAMP1extracellular domain without Peptide Signal, followed by C-terminal tagincluding 6 amino acid His-sequence.

SEQ ID NO: 30 shows the sequence of a human and mouse LAMP1 chimercontaining mouse Loop1 region of LAMP1 and human Loop2-4 of LAMP1without Peptide Signal, followed by C-terminal 6 amino acid His-Tag.

SEQ ID NO: 31 shows the sequence of a human and mouse LAMP1 chimercontaining mouse Loop1-2 region of LAMP1 and human Loop3-4 of LAMP1without Peptide Signal, followed by C-terminal 6 amino acid His-Tag.

SEQ ID NO: 32 shows the sequence of a human and mouse LAMP1 chimercontaining human Loop1-2 region of LAMP1 and mouse Loop3-4 of LAMP1without Peptide Signal, followed by C-terminal tag including 6 aminoacid His sequence.

SEQ ID NO: 33 shows the sequence of a human and mouse LAMP1 chimercontaining human Loop1-3 region of LAMP1 and mouse Loop4 of LAMP1without Peptide Signal, followed by C-terminal tag including 6 aminoacid His sequence.

SEQ ID NO: 34 shows the sequence of mouse LAMP1 extracellular domainwithout Peptide Signal, followed by C-terminal tag including 6 aminoacid His sequence.

SEQ ID NO: 35 shows the light chain sequence of the “MAb1” antibody.

SEQ ID NO: 36 shows the heavy chain sequence of the “MAb1” antibody.

SEQ ID NO: 37 shows the light chain sequence of the “MAb2” antibody.

SEQ ID NO: 38 shows the heavy chain sequence of the “MAb2” antibody.

SEQ ID NO: 39 shows the predicted full-length LAMP1 protein sequence ofMacaca fascicularis.

SEQ ID NO: 40 shows the sequence of human LAMP2 extracellular domainwithout Peptide Signal, followed by C-terminal 10 amino acid His-Tag.

SEQ ID NO: 41 shows the full-length protein sequence of human LAMP2.

SEQ ID NO: 42 shows the VH sequence of the “MAb3” antibody.

SEQ ID NO: 43-45 show the sequences of the CDR1-H, CDR2-H, CDR3-H of the“MAb3” antibody.

SEQ ID NO: 46 shows the VL sequence of the “MAb3” antibody.

SEQ ID NO: 47 and 48 show the sequences of the CDR1-L and CDR3-L of the“MAb3” antibody.

SEQ ID NO: 49 shows the sequence of the heavy chain of the chimericantibody “chMAb3” antibody.

SEQ ID NO: 50 shows the sequence of the light chain of the chimericantibody “chMAb3” antibody.

SEQ ID NO: 51 shows the sequence of the variable domain of light chainof antibody “MAb3 VL_R24_R93”.

SEQ ID NO: 52 shows the sequence of CDR3-L of antibody “MAb3VL_R24_R93”.

SEQ ID NO: 53 shows the VH1 sequence of the humanized antibody“huMAb1_1” antibody.

SEQ ID NO: 54 shows the VH2 sequence of the humanized antibody“huMAb1_2” antibody.

SEQ ID NO: 55 shows the VH3 sequence of the humanized antibody“huMAb1_3” antibody.

SEQ ID NO: 56 shows the VL1 sequence of the humanized antibody“huMAb1_1” antibody.

SEQ ID NO: 57 shows the VL2 sequence of the humanized antibody“huMAb1_2” antibody.

SEQ ID NO: 58 shows the VL3 sequence of the humanized antibody“huMAb1_3” antibody.

SEQ ID NO: 59 shows the light chain variant 1 sequence of the “huMAb1_1”antibody.

SEQ ID NO: 60 shows the heavy chain variant 1 sequence of the “huMAb1_1”antibody.

SEQ ID NO: 61 shows the light chain variant 2 sequence of the “huMAb1_2”antibody.

SEQ ID NO: 62 shows the heavy chain variant 2 sequence of the “huMAb1_2”antibody.

SEQ ID NO: 63 shows the light chain variant 3 sequence of the “huMAb1_3”antibody.

SEQ ID NO: 64 shows the heavy chain variant 3 sequence of the “huMAb1_3”antibody.

SEQ ID NO: 65 shows the light chain sequence of the negative control“huMAb1_negA” antibody with the mutations 36A and 95A.

SEQ ID NO: 66 shows the heavy chain sequence of the negative control“huMAb1_negA” antibody with the mutation 101A.

SEQ ID NO: 67 shows the heavy chain sequence of the negative control“huMAb1_negB” antibody with the mutation 266A.

SEQ ID NO: 68 shows the light chain sequence of the recombinant huMAb1_1for cristalization.

SEQ ID NO: 69 shows the heavy chain sequence of the recombinant huMAb1_1for cristalization comprising a C-terminal His-tag.

SEQ ID NO: 70 shows the sequence of a human Loop1-2 region of LAMP1 witha cleavable thioredoxin (trx A) tag, a His-Tag and a thrombin cleavagesite.

SEQ ID NO: 71 shows the sequence of the untagged hLAMP1-29-195.

SEQ ID NO: 72 shows the amino acid sequence corresponding to the aminoacids 101 to 110 of SEQ ID NO: 24.

SEQ ID NO: 73 shows the amino acid sequence corresponding to the aminoacids 144 to 157 of SEQ ID NO: 24.

SEQ ID NO: 74 shows the amino acid sequence corresponding to the aminoacids 174 to 188 of SEQ ID NO: 24.

SEQ ID NO: 75 shows the amino acid sequence corresponding to the aminoacids 29 to 41 of SEQ ID NO: 24.

SEQ ID NO: 76 shows the amino acid sequence corresponding to the aminoacids 68 to 80 of SEQ ID NO: 24.

SEQ ID NO: 77 shows the amino acid sequence corresponding to the aminoacids 29 to 100 of SEQ ID NO: 24.

SEQ ID NO: 78 shows the amino acid sequence corresponding to the aminoacids 97 to 110 of SEQ ID NO: 24.

SEQ ID NO: 79 shows the amino acid sequence corresponding to the aminoacids 173 to 189 of SEQ ID NO: 24.

SEQ ID NO: 80 shows the amino acid sequence corresponding to the aminoacids 132 to 302 of SEQ ID NO: 70.

SEQ ID NO: 81 shows the sequence of the light chain of the chimericantibody “chMAb3 VL_R24_R93”. SEQ ID NO: 82 shows the amino acidsequence corresponding to the amino acids 360 to 375 of SEQ ID NO: 24.

SEQ ID NO: 83-85 show the sequences of the CDR1-H, CDR2-H, CDR3-H of the“MAb4” antibody.

SEQ ID NO: 86 and 87 show the sequences of the CDR1-L and CDR3-L of the“MAb4” antibody.

SEQ ID NO: 88 shows the VH1 sequence of the antibody “MAb4”.

SEQ ID NO: 89 shows the VL1 sequence of the antibody “MAb4”.

SEQ ID NO: 90 shows the amino acid sequence corresponding to the aminoacids 47 to 61 of SEQ ID NO: 24.

SEQ ID NO: 91 shows the amino acid sequence corresponding to the aminoacids 140 to 155 of SEQ ID NO: 24.

SEQ ID NO: 92 shows the amino acid sequence corresponding to the aminoacids 307 to 321 of SEQ ID NO: 24.

SEQ ID NO: 93 shows a consensus sequence for CDR1-L ofMAb1/huMAb1_1/huMAb1_2/huMAb1_3 antibody family based on residuesidentified as important for the canonical structure and thus the bindingof human LAMP1 using cristallography.

SEQ ID NO: 94 shows a consensus sequence for CDR3-L ofMAb1/huMAb1_1/huMAb1_2/huMAb1_3 antibody family based on residuesidentified as important for the canonical structure and thus the bindingof human LAMP1 using cristallography.

SEQ ID NO: 95 shows a consensus sequence for CDR1-H ofMAb1/huMAb1_1/huMAb1_2/huMAb1_3 antibody family based on residuesidentified as important for the canonical structure and thus the bindingof human LAMP1 using cristallography.

SEQ ID NO: 96 shows a consensus sequence for CDR3-H ofMAb1/huMAb1_1/huMAb1_2/huMAb1_3 antibody family based on residuesidentified as important for the canonical structure and thus the bindingof human LAMP1 using crystallography.

SEQ ID NO: 97 shows the amino acid sequence corresponding to the aminoacids 35 to 84 of SEQ ID NO: 24.

SEQ ID NO: 98 shows the light chain sequence of the “MAb4” antibody.

SEQ ID NO: 99 shows the heavy chain sequence of the “MAb4” antibody.

Examples Example 1: Preparation of Patient-Derived Tumor Xenografts(PDX) Example 1.1: Preparation of CR-LRB-010P, CR-LRB-003P, andCR-IGR-034P PDXs

A large collection of colorectal cancer models directly derived fromtumor samples collected during patient surgery was develop.Patient-derived colorectal cancer tumor were collected, after patient'sinformed consent, in 3 medical centers: Curie Institute (Paris, France),Gustave Roussy Institute (Villejuif, France), and Lariboisiere Hospital(Paris, France). Immediately after surgery (1 hour after resection inaverage), 2 fragments were transferred in culture medium including DMEMwith 10 mmol/L HEPES, 4.5 g/L glucose, 1 mmol/L pyruvate sodium, 200U/mL penicillin, 200 mg/mL streptomycin, 200 mg/mL gentamicin, 5 mg/mLciprofloxacin, 20 mg/mL metronidazole, 25 mg/mL vancomycin, and 2.5mg/mL fungizone or DMEM with Nanomycopulitine (Abcys) for engraftment.After 2 to 24 hours following the patient surgery, the tumor sampleswere engrafted on 2 Swiss nude mice. Small fragments (50 mm³) weresubcutaneously engrafted into the scapular area or on the flank ofanesthetized mice. (xylazine/ketamine or isoflurane protocol). Tumorgrowth was measured at least once a week and serial fragment grafts ofeach given tumor were conducted on 3 to 5 Swiss nude or CB17-SCID (after3 passages) mice when the tumors reached a volume of 800 to 1500 mm³.(Julien, S. 2012, Clin. Cancer Res. 18(19):5314-5328.

Example 1.2: Preparation of LUN-NIC-0014 PDX and LUN-NIC-0070 PDXs

Non-small-cell lung carcinoma samples were collected, after patient'sinformed consent, in CHU Pasteur (Nice, France). Immediately aftersurgery, a piece of the patient tumor was transferred in AQIX medium andsent to Sanofi (Vitry sur Seine, France). After 24 to 48 hours followingthe patient surgery, the tumors samples were engrafted on 2-5 CB17-SCIDmice. Small fragments (50 mm³) were subcutaneously engrafted on the miceflank. Tumor growth was followed at least once a week and serialfragment grafts of each given tumor were conducted on 5 to 10 CB17-SCID(after 3 passages) mice when the tumor reached a volume of 800 to 1500mm³.

Example 1.3: Preparation of BRE-IGR-0159 PDX

Breast carcinoma samples were collected, after patient's informedconsent, in Gustave Roussy Institute (Villejuif, France). Immediatelyafter surgery (1 hour after resection in average), 4 fragments weretransferred in culture medium including DMEM, penicillin, streptomycinand fungizone for engraftment. After a maximum of 12 hours following thepatient surgery, the tumor samples (fragments about 50 mm³) wereengrafted on fat pad on 4 BALB nude mice. Tumor growth was followed atleast once a week and sent to Sanofi (Vitry sur Seine). Serial fragmentgrafts of each given tumor were conducted on 3 to 5 BALB nude orCB17-SCID mice (after 3 passages) when the tumors reach a volume of 800to 1500 mm³.

Example 2: Generation of Monoclonal Mouse Anti LAMP1 Antibodies andFirst Screening

Immunizations, fusion and screening were performed essentially asdescribed previously using primary disaggregated tumor CR-LRB-010P orCR-LRB-003P or LUN-NIC-0014 mentioned in example 1 for immunization andP3X63-Ag8.653 myeloma cells for fusion. Using the classical methoddescribed by Wennerberg A. E et al. (1993, Am. J. Pathol. 143(4):1050-1054), 6-8 weeks old female BALB/c mice (S082342; Charles RiverLabs, Bar Harbor, Me.) each received three rounds of immunization over acourse of 41 days. Antigens were administered intraperitonealy toventral site of mice. Three days after the last injection, mice weresacrificed and spleens were isolated aseptically and washed with freshRPMI medium. Lymphocytes were released from the spleens and single-cellsuspension was washed twice with RPMI medium before being fused withP3X63-AG8.653 myeloma cells using polyethylene glycol. After fusion, thecell mixture was incubated in an incubator at 37° C. for 16-24 hours.The resulting cells preparation was transferred into selectivesemi-solid medium and aseptically plated out into 100 mm Petri platesand incubated at 37° C. Ten days after initiation of selection, theplates were examined for hybridoma growth, and visible colonies werepicked-up and placed into 96-well plates containing 200 μL of growthmedium. The 96-well plates were kept in an incubator at 37° C. for 2 to4 days.

Primary screening for IgG production was performed by Enzyme-linkedimmunosorbent assay (ELISA) using a anti-mouse kappa light chainantibody (Bethyl #A90-119A) as capturing antigen. Plates were coatedwith mouse kappa light chain antibody at 0.5 μg/well in PBS and 100μL/well of primary antibody was added to the plate. The plate wasincubated at 37° C. for 1 h and washed five times with PBS containing0.05% Tween-20 (PBS-T). Then, 100 μL of a 1:50 000 dilution of goatanti-mouse IgG (Fc) conjugated with horseradish peroxidase (Pierce#31349) was added to each well. Following incubation at 37° C. for 1 hin darkness, plates were washed with PBS-T five times. Antibody bindingwas visualized by adding TMB-H₂O₂ buffer and read at a wavelength of450. Antibodies with the murine IgG, C kappa isotype were selected forfurther screening.

Example 3: Hybridoma Screening by Immunohistochemistry (IHC)

Individual hybridoma supernatants raised against tumor tissueCR-LRB-010P were screened by IHC on a macroarray slide containing frozensections of immunizing tumor (CR-LRB-010P), human non-tumoral colon andhuman non-tumoral skin. Frozen-OCT (from Optimal Cutting Temperature)specimens of non-tumoral colon and skin were obtained from surgicalcases (commercial sources such as Asterand, US Biomax, StrasbourgHospital). The automated immunostaining was performed unsing VentanaDiscovery and Discovery XT automated systems (Ventana Medical Systems,Inc, USA).

Frozen 10 μm cryostat sections were incubated with IgG culturesupernatants as primary antibody (unknown concentration, dilution 1/3 inPhosphate Buffer Saline, PBS) for 40 min at 37° C. Culture medium wasused as negative control. A postfixation step with glutaraldehyde (0.05%in NaCl 0.9% w/v) for 4 min was done. The secondary antibody Affinipurerabbit anti-mouse IgG (315-005_008, Jackson Immunosearch Laboratories,Inc. USA) was used at 4.8 μg/mL and incubated for 12 min at 37° C.Immunostaining was done with UltraMap Red chromogenic detection kitaccording to manufacturer's recommendations for 8 min. Cryostat sectionswere subsequently counterstaining with hematoxylin II (790-2208, VentanaMedical Systems, Inc USA) and bluing for 4 min (760-2037). Stainedslides were dehydrated and coverslipped with Coverquick 2000 mountingmedium (Labonord, Ref 05547530).

Sections immunostained with mAbs were analyzed by microscope (NikonEclipse E400). After the immunohistochemical screening clones ofinterest were identified as those with reactivity with areas of tumoralcolon cells but not normal epithelial cells of colon mucosa. MAb1antibody showed evidence of tumor-associated reactivity and werenegative on epidermal human non-tumoral cells.

Similar results were obtained with MAb2 and MAb3. Based on these IHCresults, MAb1 MAb2 and MAb3 were purified for further evaluation,including extensive IHC characterization on non-tumoral and tumoraltissues for MAb1.

Example 4: mAb Characterization

Antibodies MAb1 MAb2 and MAb3 were analysed for cell surface binding onhuman primary disaggregated colon tumor by FACS using Guava®easyCyte™8HTFlow Cytometry System.

The apparent affinity expressed as EC50 values was estimated usingBIOST@T-SPEED software.

Mouse hybridomas expressing antibody were produced into T500 flask andconditioned media collected after 7 days of growth. Antibody waspurified by passing the conditioned media through a Protein-G column,washed and eluted with Glycine/HCl 100 mM pH 2.7 buffer. The eluate wasdialyzed against PBS before sterile filtration and stored at 4° C.

Example 4.1: Apparent Affinity of Antibodies MAb 1 and MAb2 to HumanPrimary Colon Tumor PDX by Row Cytometry

Advanced human primary colon tumor CR-IGR-034P was obtained fromPatient-derived xenograft in mice. Tumor CR-IGR-034P was enzymaticallydissociated using collagenase Type IV (Invitrogen; #17104-019) anddeoxyribonuclease I (Invitrogen; #18047-019) for 1 h at 4° C. Cellviability was estimated by Viacount application using Guava® easyCyte™8HT Flow Cytometry System. For apparent affinity estimation, CR-IGR-034Ptumoral cells were coated at 40,000 cells/well on 96-well High Bindplate (MSD L15X13-3) and 100 μL/well of antibody was added in 2-foldserial dilutions starting at 20 μg/ml up to 12 dilutions in assaydiluant for 45 min at 4° C. and washed three times with PBS 1% BSA. 100μL/well of goat anti-mouse IgG conjugated with Alexa647 (Invitrogen; #A2135) or goat anti-human IgG conjugated with Alexa488 (Invitrogen; #A11013) was added for 45 min at 4° C. and washed three times with PBS 1%BSA. The antibody binding was evaluated after centrifugation andresuspension of cells by adding 200 μl/well PBS 1% BSA and read usingGuava® easyCyte™ 8HT Flow Cytometry System. EC50 values were estimatedusing BIOST@T-SPEED software. EC50 values obtained with the advancedhuman primary colon tumor CR-IGR-034P are listed in Table 3.

TABLE 3 EC₅₀ obtained with CR-IGR-034P MAb1 MAb2 MAb3 CR-IGR-034P 5 nM14 nM 6 nM

Antibody binding capacity of antibody was determined using Mouse IgGCalibrator kit (Biocytex #7208) or Human IgG Calibrator Kit (Biocytex#CP010) according to the manufacturer's instructions. Antibody bindingcapacity of 230 000 and 180 000 were measured for antibody MAb1 and MAb2respectively on CR-IGR-034P.

Example 4.2: The Antibodies Bind to Multiple Cancer Cells

MAb1 and MAb2 antibodies bind to multiple cancer cells and determinationof antibody binding capacity

Antibodies were found to be able of binding to multiple tumor cells byFlow Cytometry using the conditions described in example 4.1. The panelof tumor cells comprises Patient-derived tumor xenografts from differentorigins and tumor cell lines. FIG. 2 illustrates the expression profileand Table 4 summarizes the antibody binding capacity results.

TABLE 4 Antibody Binding Capacity by FACS on Patient-derived xenograftsAntibody Binding Capacity (ABC) MAb2 MAb1 PDX/originCR-LRB-003P/colorectal 22 000 25 000 CR-LRB-010P/colorectal 95,000140,000  CR-IGR-034P/colorectal 180,000  230,000  OVA-IGR-0022/ovary60,000 67,000 STO-IND-006/stomach 64,000 90,000 LUN-NIC-025/lung 27,00033,000 LUN-NIC-014/lung 102,000  104,000  Cell lines/originColo205/colon  4,000  6,000 SW480/colon  1,700  2,500 LS174T/colon 3,600  6,000

The monoclonal antibodies MAb1 and MAb2 led to high ABC in several PDXsof colorectal, ovary, stomach and lung origin and lower ABC in celllines than in PDXs of colon origin.

MAb3 Antibodies Bind to Multiple Cancer Cells

Advanced human primary tumors from colon (CR-IGR-034P), lung(LUN-NIC-014P and breast (BRE-IGR-0159) indications were obtained frompatient-derived xenograft (PDX) in mice as described in example 1. PDXswere enzymatically dissociated using collagenase Type IV (Invitrogen;#17104-019) and deoxyribonuclease I (Invitrogen, #18047-019) for 1 h at4° C. Cell viability was estimated by Viacount application using Guava®easyCyte™ 8 HT Flow Cytometry System. Tumoral cells were coated at40,000 cells/well on 96-well High Bind plate (MSD L15XB-3) and 100 μL ofantibody was added at 20 μg/mL for 45 min at 4° C. and washed threetimes with PBS 1% BSA. 100 μL of goat anti-human IgG conjugated withAlexa488 (Invitrogen; #A11013) was added for 45 min at 4° C. and washedthree times with PBS 1% BSA. The antibody binding was evaluated aftercentrifugation and resuspension of cells by adding 200 μL/well PBS 1%BSA and read using Guava® easyCyte™ 8 HT Flow Cytometry System. The meanfluorescence was recorded and plotted in the graph shown in FIG. 12 toillustrate the expression profile of the three mAbs onto the three PDXs.Results presented in FIG. 12 show that MAb3 binds to the differentpatient-derived xenografts from colon, lung and breast origin as Mab1and Mab2 do.

Example 4.3: Internalization Score of MAb1, MAb2 and MAb3 FollowingBinding to Colon Colo205 Tumoral Cells Expressing LAMP1 by ImageStreamMultispectral Imaging Flow Cytometer (Amnis Corp.)

Viable Colo205 cells (5×10⁵ cells) were seeded into wells of 6-wellplates and incubated for 4 hours at 37° C./5% CO₂ (or 4° C. on ice fornegative control) with 10 μg/ml of AlexaFluor488-labeled antibody MAb1or AlexaFluor488-labeled antibody MAb2 or AlexaFluor488-labeled antibodyMAb3. Cells were washed by centrifugation with PBS 1% BSA at 400 g for 5minutes. Cells were fixed and permeabilized using 100 μL of Perm/Fixbuffer on ice for 20 minutes. Cells were washed by centrifugation with 1mL of Perm/Wash Cell buffer at 400 g for 5 minutes.

To test whether internalized antibodies accumulate in lysosomes,simultaneous uptake of mAbs and AlexaFluor647-labeled CD107a (alysosomal marker) were carried out. Labelled AlexaFluor647 anti-CD107aantibody at 10 μg/mL was incubated on ice for 20 minutes. Afterincubation, 1 mL Perm/Wash Cell buffer was added to wash, beforecentrifuging (400 g, 5 min). The supernatant was flicked from the platebefore the cells were fixed with 200 μL 1% formaldehyde on ice for 20minutes. The fluorescence of cells was analyzed with the ImageStreammultispectral imaging flow cytometer (Amnis corp.) using theInternalization feature. Five thousand events were acquired for eachexperimental condition and the corresponding images were analyzed usingthe IDEAS image-analysis software.

TABLE 5 Internalization score by Fluorescence-Based ImageStream ImagingFlow Cytometer Internalization score (IS) Internalization score (IS) mAb4° C., 4 hr 37° C., 4 hr MAb1 0.22 2.22 MAb2 0.19 2.24 MAb3 0.11 1.56

The monoclonal antibodies MAb1 MAb2 and MAb3 led to high internalizationscores in Colo205 cell line as shown in Table 5.

Example 4.4: Quenching of Alexa488 by Use of the Anti-Alexa488 Antibody,Flow Cytometry and Calculation of Internalized Fraction of MAb1

Alexa488-labelled MAb1 (66 nM) was incubated with 6×10⁵ Colo205 cells incomplete medium for 4 h at 37° C. or 4° C. The cells were washed twicein ice cold PBS in a cold centrifuge, and resuspended in 500 nMquenching anti-Alexa488 antibody diluted in ice cold PBS. All tubes wereincubated for 1 h on ice. Without washing, all cells were fixed in twovolumes of 2% paraformaldehyde for 10 min at room temperature. Theparaformaldehyde was removed by one wash in PBS, and the cells wereresuspended in PBS and analyzed in a flow cytometer (Guava® easyCyte 8HTFlow Cytometry System).

An internalization positive control experiment was done in parallel withAlexa488-labelled Transferrin (600 nM).

Mean fluorescence intensity (MFI) values obtained from the flowcytometry reading of 5×10⁴ cells per tube were used for allcalculations. Internalization was calculated as the MFI value ofquenched cells (intracellular compartments only) divided by the MFIvalue of unquenched cells (both cell surface and intracellularcompartments) at 37° C. as described in the formula:

${Percentage}\mspace{14mu} {of}\mspace{14mu} {internalized}\mspace{14mu} {fraction} \text{:}\; \frac{{FL}\mspace{14mu} {of}\mspace{14mu} {quenched}\mspace{14mu} {cells}\mspace{14mu} {at}\mspace{14mu} 37{^\circ}\mspace{14mu} {C.}}{{FL}\mspace{14mu} {of}\mspace{14mu} {unquenched}\mspace{14mu} {cells}\mspace{14mu} {at}\mspace{14mu} 37{^\circ}\mspace{14mu} {C.}} \times 100$

The cells incubated with Alexa488-labelled compounds at 4° C. were usedas a control since internalization of antibodies does not take placesignificantly at 4′C.

After 4 h at 37° C., about 97.0% of the total cell fluorescence fromAlexa488-MAb1 was intracellular. By comparison, about 98.5% of the totalcell fluorescence from Alexa488-Transferrin was intracellular.Transferrin is known to be internalized very efficiently by Colo205cells.

After quenching, the fluorescence of Alexa488-MAb1 measured from cellslabelled at 37° C. (both cell surface and intracellular compartments)was 10-fold higher than that of cells labelled at 4° C. (cell surface).Because the fluorescence of Alexa488-MAb1 measured at cell surface at 4°C. is proportional to the antigen density, all the above results takentogether indicate that each LAMP1 molecule is involved in several (10 onaverage) internalization cycles via recycling at cell membrane duringthe course of the experiment.

Our results show for the first time that LAMP1 can function as areceptor mediating the internalization of antibodies very efficientlyvia receptor recycling to the cell surface and suggest that theavailability of specific internalizing antibodies should aid indeveloping novel therapeutic methods to target toxins, drugs orshort-range isotopes to be delivered specifically to the interior of thecancer cells, as shown in Table 6.

TABLE 6 Internalization measurements by Flow Cytometry 4° C. 37° C. mAbQuencher 37° C. Quencher MFI, Alexa488-MAb1 4.46 172.14 167.08 MFI,Alexa488-Transferrin 8.98 1210

Example 4.5: Purification and Identification of the MAb1, MAb2 and MAb3Antibody Antigen Target

The antigen target of MAb1, MAb2 and MAb3 are purified from a membranefraction enriched by human primary colon tumor CR-LRB-010P orCR-IGR-034P using Pierce Classic IP Kit (#26146) according to themanufacturer's instructions.

Pulled-down proteins were separated by SDS-PAGE and proteins stainedwith silver nitrate. Stained bands were submitted to an in-gel trypticdigestion, and eluted peptides were analyzed by tandem MS (LC-MS/MS) onan Orbitrap bentchtop mass spectrometer (Thermo). Raw MS/MS dataanalysis with Mascot (Matrix Science) database search engine, revealedLAMP1.

This target was confirmed by ELISA with the recombinant human LAMP1 asdescribed in example 6.2 (SEQ ID NO: 28). The obtained EC₅₀ are listedin Table 7 and Table 11.

TABLE 7 EC₅₀ determined by ELISA values on recombinant human LAMP1(29-382 of SEQ ID NO: 28) Antibody EC₅₀ MAb1 0.18 nM MAb2 0.25 nM

Example 4.6: Specificity to LAMP1

LAMP2 is the closest member of the LAMP family with 35% sequenceidentity to LAMP1. For evaluating specificity to LAMP1 of MAb1, MAb2 andMAb3 antibodies, 96-well plates were coated with recombinant human LAMP2with a C-terminal 10 His-tag (SEQ ID NO: 40) (R&D Systems 6228-LM) usingthe same coating conditions described previously. Anti-LAMP1 antibodieswere added to the plates and detected by using rabbit anti-mouse IgGconjugated with horseradish peroxidase (Sigma; #A9044). Antibody bindingwas visualized by adding TMB-H₂O₂ buffer and read at a wavelength of 450nm. No binding to LAMP2 was detected with MAb1, MAb2 and MAb3antibodies.

Example 4.7: Cross-Reactivity with Cynomolgus Monkey LAMP1

Antibody MAb1 was assessed for its ability to bind primate LAMP1 proteinby ELISA. Extracellular domain of LAMP1 of human (Ala29-Met382 of SEQ IDNO: 24) and cynomolgus monkey LAMP1 (Ala27-Met380 of SEQ ID NO: 39) wereprepared as described in example 6.2. Plate was coated with cynomolgusmonkey LAMP1 protein (SEQ ID NO: 29), antibody MAb1 was added to theplate and detected with rabbit anti-mouse IgG conjugated withhorseradish peroxidase (Sigma; #A9044). The antibody binding wasvisualized by adding TMB-H₂O₂ buffer and read at a wavelength of 450 nm.Binding affinity was in the same range with both proteins as shown onFIG. 3 for MAb1.

Antibody MAb1 was also assessed for its ability to bind human LAMP1 andprimate LAMP1 proteins expressed at the surface of recombinant HEK293cells by FACS. LAMP1 Coding DNA Sequence, RefSeq NM_005561.3 (SEQ ID NO:23) was cloned internally. The CDS of Macaca mulatta LAMP1, RefSeqXP_001087801 (SEQ ID NO: 27) was also cloned internally. The predictedsequences of mature LAMP1 from Macaca mulatta and Macaca fascicularisare identical to 99%, said sequence differing by one additional Leucinat position 11 of Macaca mulatta (SEQ ID NO: 27), i.e. in the signalpeptide. The mature LAMP1 proteins of Macaca mulatta and Macacafascicularis are identical. Therefore the secreted LAMP1 used in thefollowing example is referred to cynomolgus monkey. Both CDS were clonedinto mammalian expression plasmids under CMV enhancer/promoter and SV40polyA signals. HEK293 cells (Invitrogen; #K9000-10.) were transientlytransfected with human LAMP1 or cynomolgus LAMP1 plasmids usingFreeStyle™ MAX 293 Expression System according to the manufacturer'sinstructions. Human LAMP1 transfected HEK293 cells and cynomolgus LAMP1transfected HEK293 cells were coated at 40,000 cells/well on 96-wellHigh Bind plate (MSD L15XB-3) and 100 μL/well of antibody MAb1 was addedin 2-fold serial dilutions starting at 20 μg/ml up to 12 dilutions inassay diluant for 45 min at 4° C. and washed three times with PBS 1%BSA. 100 μL/well of goat anti-mouse IgG conjugated with Alexa647(Invitrogen; # A2135) was added for 45 min at 4° C. and washed threetimes with PBS 1% BSA. The antibody binding was evaluated aftercentrifugation and resuspension of cells by adding 200 μl/well PBS 1%BSA and read using Guava® easyCyte™ 8HT Flow Cytometry System. EC50values were estimated using BIOST@T-SPEED software. Binding affinity wasin the same range with EC₅₀ of 14 and 44 nM to respectively human andcynomlogus monkey LAMP1 expressed transiently at the cell surface ofHEK293 for MAb1.

Antibody MAb1 was assessed for its ability to bind human LAMP1 andprimate LAMP1 proteins expressed at the surface of recombinant HCT116stable clones by FACS. HCT116 cells were infected by a lentiviral vectorallowing stable integration of the human or the cynomolgus LAMP1 CDS ingenomic DNA of cells. Individual clones with different densities ofhuman or cynomolgus LAMP1 cell surface localization were derived from apool of HCT116 infected cells. HCT116 cells expressing human orcynomolgus LAMP1 were plated in 96-well plates at 200 000 per well andMAb1 was added in 2-fold serial dilutions starting at 40 μg/ml up to 12dilutions in assay diluant for 1 h at 4° C. and washed two times withPBS 1% BSA. 100 μL/well of goat anti-human IgG conjugated with Alexa488(Invitrogen; # A11013) was added for 1 h at 4° C. and washed two timeswith PBS 1% BSA. The antibody binding was evaluated after centrifugationand resuspension of cells in 100 μl fixing solution (paraformaldehyde at4% in PBS). Samples were read using Galaxy® Flow Cytometry System(Partec). EC50 values were estimated using BIOST@T-SPEED software.Antibody MAb1 binds to human and cynomolgus LAMP1 expressed at the cellsurface of recombinant HCT116 with similar affinity and EC₅₀ of 4.9 and5.5 nM respectively.

Antibody MAb2 was assessed for its ability to bind human LAMP1 andprimate LAMP1 proteins expressed at the surface of recombinant HCT116stable clones by FACS. Recombinant HCT116 cells were coated at 40,000cells/well on 96-well High Bind plate (MSD L15XB-3) and 100 μL/well ofantibody MAb2 was added in 2-fold serial dilutions starting at 20 μg/mlup to 12 dilutions in assay diluant for 45 min at 4° C. and washed threetimes with PBS 1% BSA. 100 μL/well of goat anti-mouse IgG conjugatedwith Alexa647 (Invitrogen; # A2135) was added for 45 min at 4° C. andwashed three times with PBS 1% BSA. The antibody binding was evaluatedafter centrifugation and re-suspension of cells by adding 200 μl/wellPBS 1% BSA and read using Guava® easyCyte™ 8HT Flow Cytometry System.EC50 values were estimated using BIOST@T-SPEED software. Antibody MAb2binds to human and cynomolgus LAMP1 expressed at the cell surface ofrecombinant HCT116 with similar affinity and EC₅₀ of 6.3 and 6.6 nMrespectively for MAb2.

Therefore MAb1 and MAb2 bind to LAMP1 of human and cynomolgus originwith similar affinity.

Antibody MAb3 was assessed by flow cytometry for its ability to bind tohuman LAMP1 and primate LAMP1 proteins expressed respectively at thesurface of HCT116 or HEK293 stable clones. HCT116 stable clone wasobtained as described above. HEK293 cells were infected by a lentiviralvector allowing stable integration of the human or the cynomolgus LAMP1CDS in genomic DNA of cells. Individual clones with different densitiesof cynomolgus LAMP1 cell surface localization were derived from a poolof HEK293 infected cells. Protocol as described in example above. EC₅₀values were estimated using BIOST@T-SPEED software. Antibody MAb3 bindsto human and cynomolgus LAMP1 expressed at the surface of HCT116 orHEK293 with similar affinity and EC₅₀ of 7.6 and 4.0 nM respectively.

Therefore MAb1, MAb2 and MAb3 bind to LAMP1 of human and cynomolgusorigin with similar affinity.

Example 4.8: Binding Competition Between MAb According to the Inventionand/or Commercially Available Anti-LAMP1 H4A3

The following examples present information on the competition of themAbs towards the epitope onto LAMP1 by ELISA. It confirmed data obtainedon the epitope binding site as described in example 6 and allowed thecomparison with a commercially available anti-LAMP1 mAb.

Binding Competition Between MAb1 and MAb2

Competition between MAb2 (murine) and MAb1 (chimeric) for binding toLAMP1 was assayed by ELISA and is illustrated on FIG. 4. No competitionwas observed.

Binding Competition Between MAb1 and MAb2 or MAb3

Competition experiments between two anti-LAMP1 mAbs were performed byELISA with recombinant human LAMP1 coated on plate (as described inexample 6.2). Briefly, two mAbs were added simultaneously atconcentrations of 0.06 and 15 mg/L, the concentration of 0.06 mg/L beingclose to the EC₅₀. MAb format was chosen so that the two mAbs haddifferent Fc domains (either human or murine). Individual measurementsof mAb binding could be performed specifically by their unique specificbinding to Fc (with Peroxidase-AffiniPure Goat Anti-Human IgG Ab, FcγFragment Specific (Jackson 109-035-098) or with Peroxidase-AffiniPureGoat Anti-Mouse IgG Ab, Fcγ Fragment Specific (Jackson 115-035-164)).Results were reported as a percentage of the value obtained from the mAbalone at the same concentration, see Table 8.

TABLE 8 Competition between chMAb1 and MAb2 or MAb3 Percentage of signalcompared mAb to mAb Sample Added concen- control ID mAbs trationSecondary Ab alone 1 chMAb1 + 0.06 mg/L Anti-Human IgG_HRP 80% MAb2 15mg/L 2 chMAb1 + 0.06 mg/L Anti-Mouse IgG_HRP 100%  MAb2 15 mg/L chMAb10.06 mg/L Anti-Human IgG_HRP 100%  chMAb1 0.06 mg/L Anti-Mouse IgG_HRP 0% MAb2 15 mg/L Anti-Human IgG_HRP  0% MAb2 15 mg/L Anti-Mouse IgG_HRP100%  3 chMAb1 + 0.06 mg/L Anti-Human IgG_HRP 80% MAb3 15 mg/L 4chMAb1 + 0.06 mg/L Anti-Mouse IgG_HRP 90% MAb3 15 mg/L MAb3 15 gm/LAnti-Human IgG_HRP  0% MAb3 15 mg/L Anti-Mouse IgG_HRP 100%  5 chMAb1 +0.06 mg/L Anti-Human IgG_HRP 10% MAb1 15 mg/L 6 chMAb1 + 0.06 mg/LAnti-Mouse IgG_HRP 90% MAb1 15 mg/L MAb1 15 mg/L Anti-Human IgG_HRP  0%MAb1 15 mg/L Anti-Mouse IgG_HRP 100% 

It was found that MAb1 does not compete with MAb2 or MAb3. Therefore theLAMP1 epitope binding site for MAb1 does not overlap with the epitopebinding sites for MAb2 or MAb3.

Binding Competition Between H4A3 and MAb1 or MAb2 or MAb3 and BetweenMAb2 and MAb3

Competition experiments between anti-LAMP1 H4A3 (BioLegend 328602) andMAb1, Mab2, or MAb3 and between MAb2 and MAb3 were performed asdescribed in above Example B4.81 Results were reported as a percentageof the value obtained from the mAb alone at the same concentration, seeTable 9.

TABLE 9 Competition between H4A3 and chMAb1 or chMAb2 or chMAb3Percentage of signal compared mAb to mAb Sample Added concen- control IDmAbs tration Secondary Ab alone 1 H4A3 + 0.06 mg/L Anti-Human IgG_HRP 96% chMAb1 15 mg/L H4A3 + 0.06 mg/L Anti-Mouse IgG_HRP  98% chMAb1 15mg/L chMAb1 15 mg/L Anti-Human IgG_HRP 100% chMAb1 15 mg/L Anti-MouseIgG_HRP  0% H4A3 0.06 mg/L Anti-Human IgG_HRP  0% H4A3 0.06 mg/LAnti-Mouse IgG_HRP 100% MAb1 + 0.06 mg/L Anti-Human IgG_HRP  96% chMAb115 mg/L MAb1 + 0.06 mg/L Anti-Mouse IgG_HRP  28% chMAb1 15 mg/L MAb10.06 mg/L Anti-Human IgG_HRP  0% MAb1 0.06 mg/L Anti-Mouse IgG_HRP 100%2 H4A3 + 0.06 mg/L Anti-Human IgG_HRP 100% chMAb2 15 mg/L H4A3 + 0.06mg/L Anti-Mouse IgG_HRP  57% chMAb2 15 mg/L chMAb2 15 mg/L Anti-HumanIgG_HRP 100% chMAb2 15 mg/L Anti-Mouse IgG_HRP  0% MAb2 + 0.06 mg/LAnti-Human IgG_HRP 100% chMAb2 15 mg/L MAb2 + 0.06 mg/L Anti-MouseIgG_HRP  9% chMAb2 15 mg/L MAb2 0.06 mg/L Anti-Human IgG_HRP  0% MAb20.06 mg/L Anti-Mouse IgG_HRP 100% 3 H4A3 + 0.06 mg/L Anti-Human IgG_HRP100% chMAb3 15 mg/L H4A3 + 0.06 mg/L Anti-Mouse IgG_HRP  11% chMAb3 15mg/L chMAb3 15 mg/L Anti-Human IgG_HRP 100% chMAb3 15 mg/L Anti-MouseIgG_HRP  0% MAb3 + 0.06 mg/L Anti-Human IgG_HRP 100% chMAb3 15 mg/LMAb3 + 0.06 mg/L Anti-Mouse IgG_HRP  15% chMAb3 15 mg/L MAb3 0.06 mg/LAnti-Human IgG_HRP  0% MAb3 0.06 mg/L Anti-Mouse IgG_HRP 100% 4 MAb3 +0.06 mg/L Anti-Human IgG_HRP  99% chMAb2 15 mg/L MAb3 + 0.06 mg/LAnti-Mouse IgG_HRP  58% chMAb2 15 mg/L MAb3 0.06 mg/L Anti-Human IgG_HRP 0% MAb3 0.06 mg/L Anti-Mouse IgG_HRP 100%

It was found that H4A3 competes with MAb3, partially competes with Mab2and does not compete with MAb1 for binding to LAMP1.

It was found that MAb2 and MAb3 partially compete for binding to LAMP1.

Example 5: Immunohistochemistry (IHC) Characterization of Purified MAb1on Human Non-Tumoral and Tumoral Tissues

The monoclonal antibody MAb1 was purified for further evaluation andantibody validation by extensive IHC characterization on non-tumoral andtumoral tissues. Therefore a large panel of human non-tumoral andtumoral tissues from commercial Tissue-Micro-Arrays or whole cryostatsections was tested for LAMP1 immunoreactivity either as Frozen-OCTs(Optimal Cutting Temperature) or Acetic Formalin Alcohol (AFA) orformalin patient-derived human xenografts. The PDXs samples used weredescribed in example 1.

Immunostaining on AFA Format

Classical IHC was performed using Ventana automatic instrument(Discovery XT, Ventana Medical Systems, Inc, USA). Sections were dewaxedand incubated with avidin and biotin blocking reagent (Endogenous Block,Ventana, 760-050) followed by Serum Block incubation (Ventana 760-4212).The murine monoclonal antibody MAb1 was then incubated at finalconcentration of 4 μg/mL during 1 hour at 37° C. A post-fixation stepwith glutaraldehyde (0.05% in NaCl 0.9% w/v) during 4 min was done. Thesecondary goat anti-mouse IgG2a-biotinilated was incubated for 12 min at37° C. (Southern Biotech, Ref 1080-08, and dilution 1/200 in Ventana'sdiluent). Immunostaining was done with DAB Map chromogenic detection kitaccording to manufacturer's recommendations. A counterstaining step wasapplied to the cryostat sections with hematoxylin II (790-2208, VentanaMedical Systems, Inc USA) and bluing reagent was applied for 4 min(760-2037). Stained slides were dehydrated and coverslipped withCoverquick 2000 mounting medium (Labonord, ref 05547530). The negativecontrols used in this study consisted in omission of primary antibodyand the use of IgG2a isotype (final concentration 1 μg/mL in PBS).

Immunostaining on PFA Format

Classical IHC was performed using Ventana automatic instrument(Discovery XT, Ventana Medical Systems, Inc, USA). Sections were dewaxedand antigen retrieval Cell Conditioning 1 (CC1) buffer (ref 950-123Ventana) was applied during 52 min. The sections were incubated withavidin and biotin blocking reagent (Endogenous Block, Ventana, 760-050)and Serum Block reagent (Ventana, 760-4212). The murine monoclonalantibody MAb1 was then incubated at final concentration of 4 μg/mLduring 1 hour at 37° C. A post-fixation step with glutaraldehyde (0.05%in NaCl 0.9% w/v) during 4 min was done. The secondary goat anti-mouseIgG2a-biotinilated was incubated for 12 min at 37° C. (Southern Biotech,Ref 1080-08, and dilution 1/200 in Ventana's diluent). Immunostainingwas done with DAB Map chromogenic detection kit according tomanufacturer's recommendations. A counterstaining step was applied tothe cryostat sections with hematoxylin II (790-2208, Ventana MedicalSystems, Inc USA) and bluing reagent was applied for 4 min (760-2037).Stained slides were dehydrated and coverslipped with Coverquick 2000mounting medium (Labonord, ref 05547530). The negative controls used inthis study consisted in omission of primary antibody and the use ofIgG2a isotype (final concentration 1 μg/mL in PBS).

Immunostaining on Frozen-OCT Format

After avidin and biotin blocking (Endogenous Block, Ventana, 760-050),frozen sections were incubated with murine monoclonal antibody MAb1(final concentration 1 μg/mL (for human samples) and 1 and 5 μg/mL (formonkey samples) in Phosphate Buffer Saline, PBS) for 32 min at 37° C. Apostfixation step with glutaraldehyde (0.05% in NaCl 0.9% w/v) for 4 minwas done. The secondary goat anti-mouse IgG2a-biotinylated was incubatedfor 12 min at 37° C. (Southern Biotech, Ref 1080-08, dilution 1/200 inVentana's diluent). Immunostaining was done with DAB Map chromogenicdetection kit according to manufacturer's recommendations. Acounterstaining step was applied to the cryostat sections withhematoxylin II (790-2208, Ventana Medical Systems, Inc USA) and bluingreagent was applied for 4 min (760-2037). Stained slides were dehydratedand coverslipped with Coverquick 2000 mounting medium (Labonord, Ref05547530).

The negative controls used in this study consisted in omission ofprimary antibody and the use of IgG2a isotype (final concentration 1μg/mL in PBS).

Data Analysis

Sections immunostained with purified murine antibody MAb1 were scannedand digitized at a magnification of ×20 using Scan Scope XT system(Aperio Technologies, Vista Calif.). Digitized images were then capturedusing Image Scope software (v10.2.2.2319 Aperio, Technologies).

Staining evaluation included several parameters: histologic site ofreactivity (cytoplasm, nuclei or membrane), main type of reactive cell,staining intensity and cell staining frequency. The positive sampleswere scored with a scale of intensity from 1 to 3. Ranges of intensitieswere described as negative (0), weak (1), moderate (2) and strong (3).Cell frequency was the percentage of immunostained cells and wasestimated by the histologist observation as a median by sample. The cellfrequency was ordered in 5 categories: 1 (0-5%), 2 (6-25%), 3 (26-50%),4 (51-75%) and 5 (76-100%).

A global expression was calculated according the Allred Score (AS)description. AS was obtained by adding the intensity and the proportionscores to obtain a total score that ranged from 0-8. The AS was reportedas a percent of the maximum global score and ranged in 5 categories:very low (0-25%), weak (26-50%), moderate (51-75%) and high (75-100%).The prevalence was defined as the percent of positive cases for theindication.

Basic descriptive statistics were calculated with Microsoft Excel 2003.For each indication, number of cases, positive cases number, prevalence,intensity score mean, frequency mean and Allred score were described.

Non-Tumoral Tissue Distribution

Globally, the experimental data show that the IHC pattern of LAMP1 oncells of non-tumoral adult tissues is predominantly cytoplasmic.

LAMP1 was expressed in the cytoplasm of a large panel of tissues,including vital organs, gastrointestinal, reproductive, urinary,endocrine, lymphoid and others as skin, muscle, eye, spinal cord) and nomembrane staining was observed in main organs as heart, liver, pancreas,lung and kidney.

However, some LAMP1 expression at the membrane occurred but wasrestricted to stomach epithelial cells, oesophageal epithelial cells,breast epithelial cells, prostate epithelial cells, testicularepithelial cells (Table 10).

Nevertheless, prevalence and mean intensities for LAMP1 expression atthe membrane of non-tumoral samples were lower than those found intumours.

TABLE 10 LAMP1 immunostaining in human non-tumoral samples- Membranepattern Non tumoral tissues % Prev Intensity % +cells Prev % Prv PrevMemb Memb Memb Tissue Type N Cyto Cyto Memb (Mean) (Mean) (Mean) Celltype Stomach 28 28/28 100% 3/28 11% 2 16 Epithelial C. Esophagus 1716/17  94% 2/17 12% 2.5 5 Epithelial Basal C. Breast 17 17/17 100% 6/1735% 1.5 15 Epithelial C. Prostate 26 26/26 100% 1/26  4% 2 5 EpithelialC. Testis 14 14/14 100% 5/14 36% 2.2 12 Germinal + Leyding

Tumoral Tissue Distribution

The immunohistochemical pattern using MAb1 or MAb2 in human tumoraltissues demonstrates that the antigen is located in the cytoplasm and/ormembrane of tumoral tissues. Protein expression data for human tumoralsamples displaying the membrane pattern show that LAMP1 antigen is notrestricted to colon adenocarcinomas. A variety of other carcinomas,including gastrointestinal tumors (small intestine, rectum, parotidgland), vital organs tumors (lung, liver, stomach, pancreas and kidney),reproductive organ tumors (breast, ovary and prostate) as well as skin,larynx and soft tissue tumors (Table 11).

TABLE 11 LAMP1 immunostaining in human tumoral samples: Membrane patternTUMORAL TISSUES Intensity % +Cells Prev % Prev Memb Memb Alred 1- 6- 26-51- 76- Organ Tumor Type N Memb Memb (Mean) (Mean) Score Neg 5% 25% 50%75% 100% >50% Colon Adenocarcinoma 86 38/86 44 2.5 30 69 56% 17% 16% 2%7%  9% Small Adenocarcinoma 1 1/1 100 3.0 30 75 100%  Intestine RectumAdenocarcinoma 14  9/14 64 3.0 21 63 36% 21% 14% 21% 7% ParotidAdenocarcinoma 3 2/3 67 2.0 18 50 33% 33% 33% Gland Lung Squamous Cell29  6/29 21 2.5 31 69 79%  3% 10% 3% 3%  6% Carc Adenocarcinoma 12  4/1233 2.5 26 69 67%  8%  8% 17% Liver Hepatocellular 2 1/2 50 2 5 38 50%50% Carc Pancreas Adenocarcinoma 18  1/18 6 2 10 50 94%  6% Kidney ClearCell Carc 9 1/9 11 3 5 50 89% 11% Breast InvDucCar 70 27/70 39 2.4 41 6861%  3% 13%  9% 7% 7% 14% InvLobCar 3 2/3 67 2.5 60 81 33% 33% 33%  33%Ovary Adenocarcinoma 21  5/21 24 3.0 15 63 76%  5%  5% 14% SerousCarcinoma 6 1/6 17 2 10 50 83% 17% Prostate Adenocarcinoma 16  4/16 253.0 43 75 75% 19% 6%  6% Stomach Adenocarcinoma 32  8/32 25 2.3 45 NA75%  3%  9% 9% 3% 13% Skin Squamous Cell 6 1/6 17 3.0 10 63 83% 17% CarcMalignant 4 1/4 25 2.0 40 63 75% 25% Melanoma Larynx Squamous Cell 5 1/520 2.0 5 38 80% 20% Carc Soft Giant cell tumor of 2 1/2 50 3.0 5 50 50%50% Tissue thigh

Tumor indications were ranked in terms of LAMP1 expression level basedon the percentage of samples displaying more than 50% of membranefrequency (positive cells).

Based on this parameter the first tumor indications were colon, rectum,lung squamous cell carcinoma, breast invasive ductal and lobularcarcinoma, stomach adenocarcinoma and prostate adenocarcinoma.

Additionally, indications displaying 25-50% of positive cells at themembrane, could be also considered as relevant indications, includingsmall intestine adenocarcinoma, parotid gland adenocarcinoma, lungadenocarcinoma, ovary adenocarcinoma, skin malignant melanoma and larynxsquamous cell carcinoma (Table 11).

Moreover, LAMP1 immunostaining was not detected at the membrane in thefollowing tumor indications: Lung small cell carcinoma (0/3), esophagussquamous cell carcinoma (0/11), cervix squamous cell carcinoma 0/3),endometrium adenocarcinoma (0/3), vulva squamous cell carcinoma (0/6),testis seminoma (0/4), testis embryonal carcinoma (0/1), bladdertransitional cell carcinoma (0/1), thyroid papillary adenocarcinoma(0/3) and mullerian mixed tumor of the oral cavity (0/5).

Example 6—Binding Site Identification

In this example LAMP1 domains were defined and human-murine hybrid LAMP1proteins were designed to generate secreted as well as membrane-anchoredLAMP1 proteins allowing the characterization of the binding site of theanti-LAMP1 mAbs towards LAMP1.

Example 6.1: Definition of LAMP1 Domains

LAMP1 also named CD107a is the Lysosomal Associated Membrane Protein 1.It is a transmembrane type I protein of around 120 kDa. The protein is ahighly glycosylated monomer with eighteen N-glycosylation and sixO-glycosylation sites. It is composed of two lumenal domains separatedby a hinge. Each lumenal domain has two disulphide bridges that definetwo loops. According to RefSeq NP_005552.3 (SEQ ID NO: 24) the differentdomains of LAMP1 have been mapped as shown in Table 1. Based onstructural information and in particular beta-strands and amino acidsdifferences between human and mouse LAMP1 several hybrid LAMP1 moleculeswere designed.

Example 6.2: Preparation of Recombinant Extracellular Domains of LAMP1Proteins

The high level of glycosylation of the antigen required a specificapproach to determine the binding site of the anti-LAMP1 mAbs on LAMP1.The LAMP1 monoclonal antibodies MAb1 and MAb2 do not show any binding tothe mouse LAMP1 protein. This absence of binding was used to designseveral chimeric LAMP1 proteins in which one or several of the LAMP1domains (Loop1-Loop4) in the human construct were replaced by the murinecounterpart. The absence of binding once the binding site of theantibody was replaced by the murine counterpart allowed foridentification of the antibody binding side.

Hence, the extracellular protein domains of LAMP1 from human, cynomolgusmonkey (c) and murine (m) origin or hybrid between murine and humanLAMP1 domains have been prepared by transient expression in humanembryonic kidney HEK293 cells with plasmids allowing expression of therespective cDNA as outlined on Table 12.

Each expression plasmid was complexed with 293Fectin™ (LifeTechnologies) and eight days post-transfection in suspension-cultivated293-F cells (derived from HEK293 cells), the corresponding solubleprotein was purified by IMAC (GE Healthcare) to generate a proteinbatch.

TABLE 12 Description of the recombinant extracellular domains of LAMP1proteins Protein name Description of protein domains Sequence ID.LAMP1::histag human LAMP1 (29-382) SEQ ID NO: 28 cLAMP1::histagcynomolgus LAMP1 (27-380) SEQ ID NO: 29 mLAMP1_L1_LAMP1_L234::histagLoop1: mouse LAMP1 (25-94) SEQ ID NO: 30 Loop2-4: human LAMP1 (101-382)mLAMP1_L12_LAMP1_L34::histag Loop1-2: mouse LAMP1 (25-189) SEQ ID NO: 31Loop3-4: human LAMP1 (196-382) LAMP1_L12_mLAMP1_L34::histag Loop1-2:human LAMP1 (29-195) SEQ ID NO: 32 Loop3-4: mouse LAMP1 (190-369)LAMP1_L123_mLAMP1_L4::histag Loop1-3: human LAMP1 (29-309) SEQ ID NO: 33Loop4: mouseLAMP1 (299-369) mLAMP1::histag mouse LAMP1 (25-369) SEQ IDNO: 34

Example 6.3: Determination of Binding Affinity and Epitope by ELISA

Secreted LAMP1 proteins described in example 6.2 were used to identifythe binding domain to anti-LAMP1 mAbs by ELISA. MAb1 recognizes loop 2of LAMP1 and MAb2 recognizes loop 1 of LAMP1 with EC₅₀ to LAMP1 ofaround 0.2 and 0.3 nM respectively.

TABLE 13 EC₅₀ (nM) obtained for murine or chimeric hybridoma mAbsProtein Loop1: Loop1-2: Loop1-2: Loop1-3: mLAMP1 mLAMP1 hLAMP1 hLAMP1Anti- human/ Mouse Loop2-4: Loop3-4: Loop3-4: Loop4: body LAMP1 LAMP1hLAMP1 hLAMP1 mLAMP1 mLAMP1 MAb1 0.18 No 0.15 No binding 0.18 0.16binding MAb2 0.25 No No No binding 0.25 0.25 binding binding chMAb1 0.12No 0.11 No binding 0.11 0.11 binding chMAb3 0.11 No No No binding 0.120.11 binding binding

Example 6.4: Expression of LAMP1 Transmembrane Proteins

Different LAMP1 proteins were expressed at the cell membrane of HEK293cells after transient expression from mammalian plasmids encoding theentire coding sequence of LAMP1 deleted of the intracellularlysosome-targeting motif GYQTI and substituted by a 5-Ala repeatsequence. Mammalian plasmids had similar expression signals as plasmidsused to produce recombinant LAMP1 described in example 6.2. Table 14below lists all the plasmids that were designed in order to confirm theresults obtained with soluble LAMP1 protein by ELISA in example 6.3 andfurther characterize the binding domains of the anti-LAMP1 mAbs.

TABLE 14 Description of LAMP1 transmembrane proteins Short descriptionof LAMP1 transmembrane protein/ Plasmid Encoded Protein with amino acidpositions according to SEQ ID NO: 24 pXL5626 hLAMP1_ΔGYQTI human LAMP1pXL5668 LAMP1_mL1_hL234_ΔGYQTI Hybrid LAMP1 murine in L1 and human in L2to L4 pXL5669 LAMP1_hL12_mL34_ΔGYQTI Hybrid LAMP1 human in L1 and L2murine in L3 and L4 pXL5719 hLAMP1_ΔglycaninL1_ΔGYQTI human LAMP1 withsubstitution of N > Q at positions 37, 45, 62, 76 and 84 in L1 pXL5720hLAMP1_ΔglycaninL2_ΔGYQTI human LAMP1 with substitution of N > Q atpositions 103, 107, 121, 130, 165 and 181 in L2 pXL5988LAMP1_mL1_hL2_mL34_ΔGYQTI Hybrid LAMP1 murine in L1, L3 and L4 human inL2 pXL5997 LAMP1_hL1_mL2_hL34_ΔGYQTI Hybrid LAMP1 human in L1, L3 and L4murine in L2 pXL5990 LAMP1_mseq6_ΔGYQTI Hybrid LAMP1; human sequenceexcept murine sequence at position 97 to 110 in L2 pXL5991LAMP1_mseq7_ΔGYQTI Hybrid LAMP1 human sequence except murine sequence atposition 110 to 128 in L2 pXL5992 LAMP1_mseq8_ΔGYQTI Hybrid LAMP1 humansequence except murine sequence at position 128 to 144 in L2 pXL5993LAMP1_mseq9_ΔGYQTI Hybrid LAMP1 human sequence except murine sequence atposition 144 to 157 in L2 pXL5994 LAMP1_mseq10_ΔGYQTI Hybrid LAMP1 humansequence except murine sequence at position 157 to 173 in L2 pXL5995LAMP1_mseq11_ΔGYQTI Hybrid LAMP1 human sequence except murine sequenceat position 173 to 189 in L2 pXL5996 LAMP1_mseq12_ΔGYQTI Hybrid LAMP1human sequence except murine sequence at position 189 to 196 in L2pXL6009 mLAMP1_ΔGYQTI murine LAMP1 pXL6012 LAMP1_mseq1_ΔGYQTI HybridLAMP1 human sequence except murine sequence at position 29 to 41 in L1pXL6013 LAMP1_mseq2_ΔGYQTI Hybrid LAMP1 human sequence except murinesequence at position 41 to 56 in L1 pXL6014 LAMP1_mseq3_ΔGYQTI HybridLAMP1 human sequence except murine sequence at position 56 to 68 in L1pXL6015 LAMP1_mseq4_ΔGYQTI Hybrid LAMP1 human sequence except murinesequence at position 68 to 80 in L1 pXL6017 LAMP1_mseq5_ΔGYQTI HybridLAMP1 human sequence except murine sequence at position 80 to 97 inL1/L2 pXL6041 mLAMP1_hseq6-11_ΔGYQTI Hybrid LAMP1 murine sequence excepthuman sequence at position 91 to 104 and 167 to 183 in L2 pXL6047 mLAMP1_hseq6_ΔGYQTI Hybrid LAMP1 murine sequence except human sequence atposition 91 to 104 in L2 pXL6048 mLAMP1_hseq11_ΔGYQTI Hybrid LAMP1murine sequence except human sequence at position 167 to 183 in L2pXL6092 mLAMP1_hseq6-9-11_ΔGYQTI hybrid LAMP1 murine sequence excepthuman sequence at position 91 to 104 and 138 to 151 and 167 to 183 in L2

Example 6.5: Determination of Binding Affinity and Epitope by FlowCytometry

Each expression plasmid described in example 6.4 was complexed with293Fectin™ in suspension-cultivated 293-F cells as using the protocoloutlined in example 6.2. Two days post transfection cells wereprocessed, analyzed by flow cytometry (Guava® easyCyte™ 8 HT) asmentioned in example 4.7, and the mean fluorescence was recorded. Thisfluorescence represents a semi-quantitative assessment of binding.

The results obtained with plasmids pXL5626, pXL5668, pXL5669, pXL5719and pXL5720 are summarized in Table 15.

TABLE 15 Binding of huMAb1_1, chMAb1, chMAb2 and chMAb3 onto LAMP1proteins by flow cytometry (Mean fluorescence) Plasmid pXL5626 pXL5668pXL5669 pXL5719 pXL5720 Protein hLAMP1_ΔGYQTI LAMP1_mL1_hL234_LAMP1_hL12_mL34_ hLAMP1_ΔglycaninL1_ hLAMP1_ΔglycaninL2_ ΔGYQTI ΔGYQTIΔGYQTI ΔGYQTI huMAb1_1 864 1112 934 1103 528 Binding Binding BindingBinding Binding chMAb1 1047 1059 1484 1113 1006 Binding Binding BindingBinding Binding chMAb2 1025 6 814 458 958 Binding No binding BindingBinding Binding chMAb3 640 21 764 706 765 Binding No binding BindingBinding Binding

This first set of affinity data (Table 15) with membrane-anchored LAMP1proteins are in agreement with the ELISA data reported on Example 6.3with the secreted LAMP1 proteins. MAb1 binds to hLAMP1 in L2 positions101 to 195 of hLAMP1 (SEQ ID No: 24), MAb2 and MAb3 bind to hLAMP1 in L1positions 29 to 100 of hLAMP1. These data also showed that none of thethree anti-LAMP1 bind to a glycotope since MAb1 binds to LAMP1 for whichL2 was engineered to have no N-glycosylation site and MAb2 and MAb3 bindto LAMP1 for which L1 was engineered to have no N-glycosylation site.The results obtained with plasmids pXL5626, pXL5988, pXL5669, pXL5990 topXL5997 are summarized in Table 16.

TABLE 16 Binding of huMAb1_1 and chMAb2 onto LAMP1 proteins by flowcytometry (mean fluorescence) Plasmid Protein huMAb1_1 chMAb2 pXL5626hLAMP1_ΔGYQTI 1412 1498 Binding Binding pXL5988LAMP1_mL1_hL2_mL34_ΔGYQTI 1180 10 Binding No binding pXL5997 LAMP1 _hL1_mL2_hL34_ΔGYQTI 25 1167 No Binding Binding pXL5990 LAMP1_mseq6_ΔGYQTI11 1721 No Binding Binding pXL5991 LAMP1_mseq7_ΔGYQTI 1400 1412 BindingBinding pXL5992 LAMP1_mseq8_ΔGYQTI 1440 1688 Binding Binding pXL5993LAMP1_mseq9_ΔGYQTI 545 1461 Binding Binding pXL5994 LAMP1_mseq10_ΔGYQTI1414 1555 Binding Binding pXL5995 LAMP1_mseq11_ΔGYQTI 16 1378 No BindingBinding pXL5996 LAMP1 mseq12_ΔGYQTI 1303 1365 Binding Binding ballast noLAMP1 1 1 No binding No binding

The results obtained with plasmids pXL5626, pXL6041, pXL6047, pXL6048,and pXL6009 are summarized in Table 17.

TABLE 17 Binding of MAb1 and MAb2 onto LAMP1 proteins by flow cytometry(mean fluorescence Experiment n° 1 Experiment n° 2 Plasmid Protein MAb1MAb2 MAb1 pXL5626 hLAMP1_ΔGYQTI 1748 1183 551 Binding binding Fullbinding pXL6041 mLAMP1_hseq6-11_ΔGYQTI 680 28 211 Binding No bindingbinding pXL6047 mLAMP1_hseq6_ΔGYQTI 7 25 3 No binding No binding Nobinding pXL6048 mLAMP1_hseq11_ΔGYQTI 6 28 2 No binding No binding Nobinding pXL6092 mLAMP1_hseq6-9-11_ΔGYQTI Not done Not done 499 Fullbinding pXL6009 mLAMP1_ΔGYQTI 4 21 2 No binding No binding No binding

The results obtained with plasmids pXL5626, pXL6012 to pXL6015, pXL6017and pXL6009 are summarized in Table 18.

TABLE 18 Binding of MAb1, MAb2 and MAb3 onto LAMP1 proteins by flowcytometry (mean fluorescence Plasmid Protein MAb1 MAb2 MAb3 pXL5626hLAMP1_ΔGYQTI 914 757 749 Binding Binding Binding pXL6012LAMP1_mseq1_ΔGYQTI 1027 105 Low 2.8 No binding binding pXL6013LAMP1_mseq2_ΔGYQTI 990 694 803 Binding Binding Binding pXL6014LAMP1_mseq3_ΔGYQTI 888 694 674 Binding Binding Binding pXL6015LAMP1_mseq4_ΔGYQTI 891 27 No 3 No Binding binding binding pXL6017LAMP1_mseq5_ΔGYQTI 846 629 721 Binding Binding Binding pXL6009mLAMP1_ΔGYQTI 13 No 27 No 3 No binding binding binding

The affinity data described in Table 16 with membrane-anchored LAMP1proteins demonstrated that MAb1 binds to LAMP1 in L2 positions 101 to195 of hLAMP1 (SEQ ID NO: 24) (pXL5626, pXL5988 and pXL5997). Morespecifically MAb1 does not bind to hybrid LAMP1 protein where humanLAMP1 residues from positions 97 to 110 or from positions 173 to 189have been substituted by murine LAMP1 residues, but it binds to hybridLAMP1 protein where human LAMP1 residues from positions 110 to 173 orfrom positions 189 to 196 in L2 have been substituted by murine LAMP1residues. Of note some binding is also lost when human LAMP1 residuesfrom position 144 to 157 are replaced by the murine LAMP1 residues. Inaddition data reported in Table 17 Experiment No 1 showed that residuesfrom positions 97 to 110 and from positions 173 to 189 aresimultaneously needed to restore some of the binding. Data reported inTable 17 experiment No 2 showed that residues from positions 91 to 104and 138 to 151 and 167 to 183 in Loop2 are simultaneously needed torestore full binding of MAb1.

From these sets of affinity data (Tables 15, 16, 17 and 18) obtained byflow cytometry with membrane-anchored LAMP1 proteins and the ELISAresults with the secreted LAMP1 proteins described in Example 6.3, thefollowing conclusions could be derived:

MAb1 binds to L2 positions 101 to 195 of LAMP1, MAb2 and MAb3 bind to L1positions 29 to 100 of LAMP1.

None of the three anti-LAMP1 bind to a glycotope since MAb1 binds toLAMP1 for which L2 was engineered to have no N-glycosylation site andMAb2 and MAb3 bind to LAMP1 for which L1 was engineered to have noN-glycosylation site.

MAb1 does not bind to hybrid LAMP1 protein where human LAMP1 residuesfrom positions 97 to 110 (SEQ ID NO: 78) or from positions 173 to 189(SEQ ID NO: 79) have been substituted by murine LAMP1 residues. But itbinds to hybrid LAMP1 protein where human LAMP1 residues from positions110 to 173 or from positions 189 to 196 in L2 have been substituted bymurine LAMP1 residues.

MAb1 interacts with amino acids located within L2 and more specificallyMAb1 interacts with amino acids located within sequences from positions101 to 110 (SEQ ID NO: 72) and/or from positions 174 to 188 (SEQ ID NO:74) and to some extent to sequence from positions 144 to 157 (SEQ ID NO:73). Therefore, we can infer from these results and from amino aciddifferences between murine and human sequences that human LAMP1 residuesamong R146, D150, K152, R106, A108, N181, S182, S183, R186 and G187 arelikely to interact with MAb1.

MAb2 interacts with amino acids located within L1 and more specificallyMAb2 interacts with amino acids located within sequences from positions68 to 80 (SEQ ID NO: 76) and to some extent within sequences frompositions 29 to 41 (SEQ ID NO: 75). Therefore, we can infer from theseresults and from amino acid differences between murine and humansequences that human LAMP1 residues among A29, M30, M32, G36, A40, S69,D70, T72, V74, L75, and R77 are likely to interact with MAb2.

MAb3 interacts with amino acids located within L1 and more specificallyMAb3 interacts with amino acids located within sequences from positions29 to 41 (SEQ ID NO: 75) and/or from positions 68 to 80 (SEQ ID NO: 76).Therefore, we can infer from these results and from amino aciddifferences between murine and human sequences that human LAMP1 residuesamong A29, M30, M32, G36, A40, S69, D70, T72, V74, L75, and R77 arelikely to interact with MAb3.

Example 6.6: Determination of Individual Amino Acid Involved in EpitopeBinding by Ala Scan

Individual residues identified in example 6.5 and not involved inβ-strand structure have been individually replaced by an alanine residuein the LAMP1 sequence derived from hLAMP1_ΔGYQTI and encoded in plasmidpXL5626 (example 6.4). A total of 21 plasmids were engineered frompXL5626 (see Table 19) and used to assay LAMP1 expression at the cellmembrane of HEK293 cells after transient transfection. Two days posttransfection cells were processed, analyzed by flow cytometry (Guava®easyCyte™ 8 HT) as mentioned in example 4.7, and the mean fluorescencewas recorded. This fluorescence represents a semi-quantitativeassessment of binding. Loss of binding is reported on Table 19 whenthere is a decrease of more than 50% of the mean fluorescence comparedto the control protein encoded from pXL5626.

TABLE 19 Loss of binding to anti-LAMP1 mAb with Alascan LAMP1transmembrane proteins Position of mutation Binding Binding Plasmid inhLAMP1_ΔGYQTI to MAb1 to MAb3 pXL5626 none pXL6058 G36A pXL6065 N37ApXL6059 G38A Binding loss pXL6060 L67A pXL6066 P68A pXL6067 S69A pXL6069D70A Binding loss pXL6072 N107A pXL6073 A108T pXL6080 T109A pXL6070I149A Binding loss pXL6085 D150A Binding loss pXL6071 K151A pXL6079Y178A pXL6074 L179A pXL6075 S180A pXL6076 N181A pXL6077 F184A pXL6081R186A Binding loss pXL6082 G187A

Loss of binding to MAb1 at positions 1149, D150 and R186 due to Alasubstitution in LAMP1 protein indicates that these positions areimportant for MAb1 binding to LAMP1.

Loss of binding to MAb3 at positions G38 and D70 due to Ala substitutionin LAMP1 protein indicate that these positions are important for MAb2binding to LAMP1.

Example 7: Determination of mAb Sequences Example 7.1: Determination ofmAb Sequences and Generation of Chimeric mAbs

The sequences of the variable domains of the mAb were retrieved from thehybridoma and cloned into an expression vector to ensure that the clonedmAbs had the same characteristics as the initial murine mAbs.

The cDNA encoding the variable domains of the monoclonal antibodies wereobtained as follows. cDNA has been retrieved and sequenced by RT-PCR(transcriptase SuperScript III from Invitrogen and polymerase Phusionfrom Finnzymes) from 100 hybridoma cells and oligonucleotides located atthe 5′-end of the cDNA encoding the variable regions and the constantdomains.

High Resolution Mass Spectrometry of Hybridoma (HRMS):

Mass spectra were obtained on a Waters Synapt G2 TOF system inelectrospray positive mode (ES+). Chromatographic conditions are thefollowing: column: UPLC MassPrep 20 μm 2.1×5 mm; solvents: A: H₂O+0.1%formic acid: B; CH₃CN+0.1% formic acid; column temperature: 80° C.; flowrate 0.2 mL/min; gradient elution (10 min): 10% B for 30 sec; from 10 to50% of B in 7 min 10 sec; 8 min: 90% B; 8 min 30 sec: 10% B; 10 min: 10%B. Samples were reduced 30 min at 37° C. in Gdn.HCL 6M/DTT 1M beforeLC/MS analysis.

The derived amino acid sequences provided information in agreement withthe data obtained on purified mAbs derived from the hybridoma byN-terminal sequencing and mass spectrometry (LC/MS) of the heavy andlight chains (LC, HC) Table 20. No identical sequences were found in thepatented sequences from GenomeQuest.

TABLE 20 Mass spectrometry analysis of anti-LAMP1 mAbs from hybridomaMass (Da) by LC/MS in silico value Clone ID Chain from batch retrievedfrom sequence MAb1 LC 23587 23587 HC (G0F) 50700 50700 MAb2 LC 2391123911 HC (G0F) 50702 50704 MAb3 LC 23725 + higher 23723 masses due toN-glycans HC (G0F) 50852 50848

The nucleic acid sequences of the variable domains VH and VL were clonedinto expression vectors in fusion with the human IgG1 or the humanCkappa constant domain coding sequences, respectively, to then generatebatches of chimeric mAbs by transient expression in 293-F cells asdescribed in Example 6.2. Batches were purified by protein A affinitychromatography (MabSelect, GE Heathcare). The eluate was dialyzedagainst PBS before sterile filtration and storage at 4° C.

Affinity to LAMP1 remained similar for murine and chimeric mAbsillustrated by the EC₅₀ obtained by ELISA with LAMP1 in Table 21.

TABLE 21 EC₅₀ (nM) obtained with LAMP1 for murine hybridoma andcorresponding chimeric mAbs obtained for murine hybridoma mAbs cloneEC₅₀ obtained for chimeric mAbs ID hLAMP1 clone ID hLAMP1 MAb1 0.18 nMchMAb1 0.12 nM (assay A) 0.12 nM (assay B) MAb2 0.25 nM chMAb2 0.12 nM(assay A) chMAb2_(can) 0.12 nM (assay A) chMAb3 0.12 nM (assay B)chMAb3VL_R24_R93 0.11 nM (assay B)

Based on the data described above, the amino acid sequences of the HCand the LC were validated.

The LC and HC sequences of MAb1 are shown in SEQ ID NO: 35 and SEQ IDNO: 36, respectively.

The LC and HC sequences of MAb2 are shown in SEQ ID NO: 37 and SEQ IDNO: 38, respectively.

The sequences for the CDR regions were deduced from the protein sequenceusing the IMGT nomenclature.

The LC and HC sequences of chMAb1 are shown in SEQ ID NO: 18 and SEQ IDNO: 17, respectively, and the LC and HC sequences of chMAb2 are shown inSEQ ID NO: 20 and SEQ ID NO: 19, respectively. The LC and HC sequencesof chMAb3 are shown in SEQ ID NO: 49 and SEQ ID NO: 50, respectively.

Of note, canonical residues have been introduced into clone MAb2 atpositions A9, L51, L58, G72 and L108 on VL and at position T116 on VHsequence, to generate MAb2_(can). The corresponding amino acid sequencesof the VH and the VL of MAb2_(can) are SEQ ID NO: 15 and SEQ ID NO: 16,respectively. The HC and LC sequences of chMAb2_(can) are shown in SEQID NO: 21 and 22, respectively.

A batch of clone chMAb2_(can) was generated in the same conditions asthe batch corresponding to clone chMAb2. This highlights that pointmutations in the FR can be made without any impact on binding but moreimportantly provide an alternative to the production process.

A batch of clone chMAb3_VLR24-R93 was generated in the same conditionsas the batch corresponding to clone chMAb3. This highlights that pointmutations in the CDR can be made without any impact on binding.

Affinity to LAMP1 by SPR:

The binding kinetics of the murine, chimer or humanized anti-LAMP1 mAbswere determined by surface plasmon resonance assay using a BIAcore 2000(BIAcore Inc., Uppsala, N.J.). Briefly, a CM5 BIAcore biosensor chip wasdocked into the instrument and activated with 70 μL of 1:1 NHS/EDC atroom temperature. A mouse anti-αhuman Fc IgG1 (BIAcore #BR-1008-39) andrabbit anti-αmurine Fc IgG1 (BIAcore #BR-1008-38) (50 μg/mL in 1 Macetate buffer, pH5) were immobilized on the activated chips in all flowcells. The immobilization was carried out at a flow rate of 10 μL/min upto saturation. The chip was then blocked by injection of 70 μL ofethanolamine-HCl, pH 8.5, followed by one wash with 3 M MgCl₂ foranti-αhuman Fc IgG1 and one wash with 10 mM Glycine-HCl pH 1.7 foranti-αmurine Fc IgG1. To measure the binding of anti-LAMP1 mAbs toLAMP1, antibodies were used at 1-5 μg/mL in BIAcore running buffer(HBS-EP). The antigen (SEQ ID NO: 28 protein produced as described inexample 6.2) was injected from 1 to 256 nM. Following completion of theinjection phase, dissociation was monitored in a BIAcore running bufferat the same flow rate for 600 sec. The surface was regenerated betweeninjections using 2×5 μL 3 M MgCl₂ (2×30 s) or anti-αhuman Fc IgG1 and1×30 μL 10 mM Glycine-HCl pH 1.7 for anti-αmurine Fc IgG1 (180 s).Individual sensorgrams were analyzed using BIAevaluation software.

Affinity to LAMP1 for the murine, chimer or humanized mAbs is reportedon Table 22. It was found to be independent of the MAb format.

MAb1 binds to LAMP1 with K_(D) ranging from 4.8 to 8.2 nM

MAb2 binds to LAMP1 with K_(D) ranging from 63.5 to 68.8 nM

MAb3 binds to LAMP1 with K_(D) ranging from 4.7 to 7.2 nM

The commercially available anti-LAMP1 mAb (H4A3 (BioLegend 328602) has asignificantly higher K_(D) and thus a lower binding efficiency thanMab1, Mab 2 and MAb 3 with a Kd of around 100 nM.

TABLE 22 Binding kinetics to LAMP1 for the murine, chimer or humanizedmAbs Mab k_(a) (M⁻¹ · s⁻¹) k_(d) (s⁻¹) K_(D) (nM) Mab1 14.8E+04 0.71E−034.8 huMAb1_1 19.1E+04 1.57E−03 8.2 chMAb2can 7.21E+04 4.96E−03 68.8 Mab26.33E+04 4.02E−03 63.5 chMAb3VL_R24_R93 17.3E+04 1.25E−03 7.2 MAb324.2E+04 1.13E−03 4.7 Murine H4A3 5.80E+04 6.09E−03 105 (BioLegend328602)

Example 7.2: Obtention and Characterisation of Humanized VariantsDerived from MAb1

In this example, humanized variants of parental murine IgG MAb1 havebeen designed in silico. The resulting huMAb1 variants were produced andprovided similar characteristics as the chimer chMAb1.

Example 7.2.1 Design of the Humanized Anti-LAMP1 huMAb1 HumanizationBased on CDR Grafting

This approach consists in the transplantation of CDRs of the parentalmurine MAb1 into relevant human FRs. The variable light and heavyregions of murine MAb1 were compared to human germline sequences fromIMGT Information system (Lefranc et al. Nucl. Acids. Res. 2009,37:D1006-D1012) to select the human light and heavy variable sequencesthat would serve as the basis of the humanized MAb1 regions (huMAb1).

The mouse light chain variable region displayed 68.8% identity over theV region and 74.7% identity within the FRs alone to the human germlinekappa light chain IGKV1-27. For the joining region, mouse J regiondisplayed 90% identity to human germline IGKJ4. Consequently human Vregion IGKV1-27 combined to human J region IGKJ4 given a globalgerminality index (identity calculated on FRs only) of 76.4% have beenselected as human acceptor sequences for humanization of the mouse MAb1light chain. This then became the basis of the humanized variant of theanti-LAMP1 MAb1 light chain, which comprised the CDRs of the murine MAb1Vk region and the FRs of the human IGKV1-27_(—) IGKJ4 regions.

The mouse heavy chain variable region displayed 65.3% identity over theV region and 70.0% identity within the FRs alone to the human heavyvariable germline IGHV1-69. For the joining region, mouse J regiondisplayed 78% identity to human heavy joining germline IGHJ4.Consequently human germline V region IGHV1-69 combined to human germlineJ region IGHJ4 given a global germinality index (identity calculated onFRs only) of 71.4% have been selected as human acceptor sequences forhumanization of the murine MAb1 heavy region. This then became the basisof the humanized variant of the anti-LAMP1 MAb1 heavy chain, whichcomprised the CDRs of the murine MAb1 Vh region and the FRs of the humanIGHV1-69_IGHJ4 regions.

However, some FRs residues are also important for the biologicalactivity of the antibody since they can impact CDRs conformation andthus antigen binding. Back mutations to murine amino acid may beintroduced at selected positions of FRs grafted antibody in order toretain the binding specificity and affinity of the parent antibody.Thus, the next step in the design process was to study the proteinsequences of the humanized variant to determine if any of these aminoacid residues were likely to alter the conformation or orientation ofthe CDRs loops. A 3D homology model of the variable regions of both themurine and the humanized antibodies were built using model antibodyframework protocol of Discovery studio 3.1 from Accelrys Software Inc.

The VL and VH sequences of the murine MAb1 were compared to the proteindatabase (PDB) (Berman et al. Nucleic Acids Research, 2000, 28:235-242).

The structure model of the antithrombotic monoclonal antibody 82D6A3with the PDB identity number 2ADF was used as template for the lightchain (96.6% identity on light chain framework) and the structure modelof IL-23 in complex with neutralizing FAB with the PDB identity number3D85 was used as template for the heavy chain (83.5% identity on heavychain framework).

In the same way, the VL and VH sequences of the humanized variant (humanFRs and murine CDR) were compared to the protein database (PDB) (Bermanet al. Nucleic Acids Research, 2000, 28:235-242). The model with the PDBidentity number 3AAZ was used as template for the light chain (86.6%identity on light chain framework) and the model with the PDB identitynumber 3KDM was used as template for the heavy chain (84.3% identity onheavy chain framework) (All PDB references refer to the PDB identitynumber as available on Nov. 26, 2013).

Both 3D homology models, the murine MAb1 and the humanized version werecompared and each amino acid substitution from mouse to human versionwere carefully looked. When the substitution of a mouse to a humanresidue was done at a position that could influence the conformation ofthe CDRs, a back mutation to the murine residue was done.

Humanization Based on Molecular Dynamic Trajectories (4D HumanizationProtocol)

A molecular dynamics (MD) simulation of the 3D homology model of themurine MAb1 (as described in section above on grafting protocol) wassubsequently performed, with constraints on the protein backbone at 500K temperature for 1.1 nanoseconds (ns) in Generalized Born implicitsolvent. 10 diverse conformations were extracted from this first MD runevery 100 picoseconds (ps) for the last 1 ns. These diverseconformations were then each submitted to a MD simulation, with noconstraints on the protein backbone and at 300 K temperature, for 2.3ns. For each of the 10 MD runs, the last 2,000 snapshots, one every ps,from the MD trajectory were then used to calculate, for each murine MAb1amino acid, its root mean square deviations (rmsd) compared to areference medoid position. By comparing the average rmsd on the 10separate MD runs of a given amino acid to the overall average rmsd ofall MAb1 murine amino acids, one decides if the amino acid is flexibleenough, as seen during the MD to be considered as likely to interactwith B-cell receptors and responsible for activation of the immuneresponse. 28 amino acids were identified as flexible in the murine MAb1antibody, excluding the CDRs and its immediate 5 Å vicinity.

The motion of the 60 most flexible murine MAb1 amino acids, during the20 ns (10×2 ns) of molecular dynamic simulation, were then compared tothe motion of the corresponding flexible amino acids of 49 human 3Dhomology models, for each of which were run the same simulations. These49 human models have been built by systematically combining arepresentative panel of 7 human light chains (namely vk1, vk2, vk3, vk4,vlambda1, vlambda2, vlambda3) with a representative panel of 7 humanheavy chains (namely vh1a, vh1b, vh2, vh3, vh4, vh5, vh6).

The vk1-vh1b combination showed the highest 4D similarity of itsflexible amino acids compared to the flexible amino acids of the murineMAb1 antibody; this model was therefore used to humanize the MAb1antibody, focusing on the flexible amino acids. For the pairwise aminoacid association between the murine MAb1 and vk1-vh1b amino acids, the 2sequences were aligned based on the optimal 3D superposition of thealpha carbons of the 2 corresponding homology models.

In addition, to improve the stability of the resulting humanized MAb1antibody, the amino acids of the light and heavy chains with lowfrequency of occurrence vs their respective canonical sequences,excluding the CDRs, are originally proposed to be mutated into the mostfrequently found amino acids (ΔΔGth>0.5 kcal/mol; (Monsellier et al. J.Mol. Biol. 2006, 362, 580-593). A first list of consensus mutations forthe LC and for the HC has been restricted to the amino acids found inthe closest human model (i.e vk1-vh1b). None of these mutations arelocated in the “Vernier” zone (Foote et al., J. Mol. Biol. 1992, 224,487-499). Other criteria are taken into account to consider theseconsensus mutations for potentially stabilizing the anti-LAMP1 MAb1antibody. These criteria are a favourable change of hydropathy at thesurface or a molecular mechanics based predicted stabilization of themutant. Stabilizing mutations reported to be successful in theliterature (Bedouelle, H. J. Mol. Biol. 2006, 362, 580-593; Steipe B. J.Mol. Biol. 1994, 240, 188-192) were considered.

Resulting Humanized VL and VH Regions

Based on both approached, the CDRs grafting and the 4D protocols, threeversions for the variable light chain (VL1, VL2 and VL3) and threeversions for the variable heavy chain (VH1, VH2 and VH3) are proposed.The particular combination of amino acid residues mutated in eachhumanized MAb1 VL and VH variants are set forth in Table 23 and Table 24respectively. The complete amino acid sequences of the humanized VH andVL domains are set forth in Table 25.

For the Variable Light Region:

The humanized VL1 variant with SEQ ID NO: 56 displays a total of 12mutations compared to mouse sequence with SEQ ID NO: 5. This variantderives from frameworks of human germline IGKV1-27_IGKJ4 sequences with6 back mutations done because they were suspected to have negativeimpact on mAb structure, CDRs conformation and therefore, on binding toits target. In addition, for amino acids at position 43 and 83, mutationin a more frequent amino acid present in IGKV1 germlines (A43 and F83)was preferred.

The humanized VL2 variant with SEQ ID NO: 57 displays 2 mutations whichderive from the direct comparison between the non-CDR most flexibleamino acids of the murine MAb1 light chain and the vk1 human light chainsequence.

The humanized VL3 variant with SEQ ID NO: 58 derives from VL2 andincludes 6 new mutations that are consensus (vk1 sequence) andpotentially stabilizing.

The particular combination of amino acid residues mutated in theindividual humanized light chains of huMAb1 thus VL of huMab1_1,huMab1_2 and huMab1_3 are set forth in Table 23.

TABLE 23 Mutations of the VL variants of the anti-LAMP1 MAb1 antibodyHuMab1_1 HuMAb1_2 HuMAb1_3 Mouse MAb1 VL (VL1) (VL2) (VL3) P9 S9 S9 L15V15 V15 V15 G17 D17 K18 R18 D38 Q38 G43 A43 R45 K45 K45 P56 S56 I58 V58V58 S72 T72 F73 L73 L73 S74 T74 T74 N77 S77 I83 F83 L103 V103

For the Variable Heavy Region:

The VH1 variant (SEQ ID NO: 53) displays a total of a total of 15residues substitution compared to the mouse sequence (SEQ ID NO: 1).This variant derives from frameworks of human germline IGHV1-69_IGHJ4sequences with 9 back mutations done because they were expected to havenegative impact on mAb structure, CDRs conformation and therefore, onbinding to its target. In addition, K74 of SEQ ID NO: 1 in vicinity ofCDRs was mutated into T to anticipate a potential problem if targeted bythe conjugation process.

The VH2 variant displays 7 mutations: 6 mutations deriving from thedirect comparison between the non-CDR most flexible amino acids of themurine MAb1 heavy chain and the vh1b human heavy chain sequence, plusmutation of K74 of SEQ ID NO: 1 into T to anticipate a potential problemif targeted by the conjugation process.

The VH3 variant derives from VH2 and includes 7 new mutations that areconsensus (vh1b sequence) and potentially stabilizing.

The particular combination of amino acid residues mutated in theindividual humanized light chains of huMAb1 thus VH of huMab1_1,huMab1_2 and huMab1_3 are set forth in Table 24.

TABLE 24 Mutations of the VH variants of the anti-LAMP1 MAb1 antibodyMouse MAb1 HuMab1_1 HuMab1_2 HuMab1_3 VH (VH1) (VH2) (VH3) Q5 V5 V5 V5L11 V11 V12 K12 A16 S16 M20 V20 K38 R38 K39 Q39 S40 A40 S61 A61 K65 Q65Q65 Q65 D66 G66 G66 K67 R67 R67 K74 T74 T74 T74 S76 T76 Q82 E82 E82 E82R85 S85 S85 S85 T87 R87 S91 T91 T91 S115 T115 T115 A118 S118 S118 S118

The resulting humanized sequences were blasted for sequence similarityagainst the Immune Epitope Data Base (IEDB) database ((PLos Biol (2005)3(3)e91) http://www.immuneepitope.org;) to ensure that none of thesequences contain any known B- or T-cell epitope listed in.

The complete amino acid sequences of the humanized VH and VL domains areset forth in Table 25.

TABLE 25 VH and VL amino acid sequencesof humanized anti-LAMP1 antibodies. VH or VL SEQ ID variant Sequence NO.huMAb1_1 QVQLVQSGAEVKKPGSSVKVSCKASGYIFTN SEQ ID VH1YNIHWVKKSPGQGLEWIGAIYPGNGDAPYSQ NO: 53 KFQGKATLTADTSTSTTYMELSSLRSEDTAVYYCVRANWDVAFAYWGQGTLVTVSS huMAb1_2 QVQLVQSGAELVKPGASVKMSCKASGYIFTNSEQ ID VH2 YNIHWVKKSPGQGLEWIGAIYPGNGDAPYSQ NO: 54KFQDRATLTADTSSSTTYMELSSLTSEDSAV YYCVRANWDVAFAYWGQGTLVSVSS huMAb1_3QVQLVQSGAELVKPGASVKMSCKASGYIFTN SEQ ID VH3YNIHWVRQAPGQGLEWIGAIYPGNGDAPYAQ NO: 55 KFQGRATLTADTSSSTTYMELSSLTSEDTAVYYCVRANWDVAFAYWGQGTLVTVSS huMAb1_1 DIQMTQSPSSLSASVGDRVTITCKASQDIDRSEQ ID VL1 YMAWYQDKPGKAPRLLIHDTSTLQSGVPSRF NO: 56SGSGSGRDYTLTISNLEPEDFATYYCLQYDN LWTFGGGTKVEIK huMAb1_2DIQMTQSPPSLSASVGGKVTITCKASQDIDR SEQ ID VL2YMAWYQDKPGKGPKLLIHDTSTLQPGIPSRF NO: 57 SGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIK huMAb1_3 DIQMTQSPSSLSASVGGKVTITCKASQDIDR SEQ ID VL3YMAWYQQKPGKGPKLLIHDTSTLQPGVPSRF NO: 58 SGSGSGRDYSLTISSLEPEDIATYYCLQYDNLWTFGGGTKLEIK

Example 7.2.2: Production and Characterization of Three HumanizedAnti-LAMP1 huMAb1 Variants

The corresponding nucleic acid sequences encoding the humanized variableVH and VL domains described in example 7.2.1 were synthesized at Geneartand cloned into expression vectors in fusion with the human IgG1 or thehuman Ckappa constant domain coding sequences, respectively, to thengenerate batches of humanized mAbs by transient expression in 293-Fcells as described in Example 6.2. The three mAbs were referredto—huMAb1_1 that contains LC1 (VL1-huCk) (SEQ ID NO: 59) and HC1(VH1-hulgG1) (SEQ ID NO: 60),

-   -   huMAb1_2 that contains LC2 (VL2-huCk) (SEQ ID NO: 61) and HC2        (VH2-hulgG1) (SEQ ID NO: 62),    -   huMAb1_3 that contains LC3 (VL3-huCk) (SEQ ID NO: 63) and HC3        (VH3-hulgG1) (SEQ ID NO: 64).

A negative control was generated and referred to as huMAb1_negA. Itcontains LCnegA (VL1_36 A-95A-huCk) (SEQ ID NO: 65) and HCnegA (VH1_101A-hulgG1) (SEQ ID NO: 67).

Another control was generated and referred to as huMAb1_negB. Itcontains LC1 (VL1-huCk) (SEQ ID NO: 59) and HCnegB (VH1-hulgG1_266 A)(SEQ ID NO: 68). The mutation 266A in the hulgG1 corresponds to theD265A mutation according to the nomenclature described by Kabat et al.,Sequences of Proteins of Immunological Interest, 5th edition, NationalInstitute of Health, Bethesda, Md., 1991. It was reported tosignificantly decrease binding to FcγRs and ADCC (Lund et al., J.Immunol., 157:4963-4969, 1996; Shields et al., J. Biol. Chem., 276(1):6591-6604, 2001).

Significant decrease in binding to FcγRI, II and III was also verifiedby ELISA with recombinant proteins (recombinant human FcγRI/CD64reference 1257-FC-050, recombinant human FcγRIIA/CD32a reference1330-CD-050/CF, recombinant human FcγRIIIa/CD16a, reference 4325-FC-050,all obtained from R&D System).

Batches were purified by protein A affinity chromatography (MabSelect,GE Heathcare). The eluate was dialyzed against PBS before sterilefiltration and storage at 4° C. Batches were analysed by High ResolutionMass Spectrometry as described in Example 7. Data were in agreement withthe in silico value retrieved from amino acid sequences, Table 26.

TABLE 26 Mass spectrometry analysis of humanized anti-LAMP1 mAbs Mass(Da) by LC/MS in silico value mAb ID Chain from batch retrieved fromsequence huMAb1_1 LC1 23483 Da 23484 Da HC1 (G0F) 50219 Da 50219 DahuMab1_2 LC2 23375 Da 23376 Da HC2 (G0F) 50209 Da 50209 Da huMAb1_3 LC323318 Da 23318 Da HC3 (G0F) 50176 Da 50175 Da huMAb1_negA LCnegA 23277Da 23277 Da HCnegA 50103 Da 50104 Da (G0F) huMAb1_negB LC1 23484 Da23484 Da HCnegB 50175 Da 50175 Da (G0F)

Secreted human LAMP1 protein described in example 6.2 was used todetermine the binding domain to the humanized anti-LAMP1 mAbs by ELISA.Affinity to LAMP1 remained similar for chimer and humanized mAbs asillustrated by the EC₅₀ obtained by ELISA with LAMP1 in Table 27. Nobinding is detected with huMAb1_negA.

TABLE 27 EC₅₀ (nM) obtained with LAMP1 for chimer and humanized mAbs mAbID hLAMP1 chMAb1 0.09 nM huMAb1_1 0.11 nM huMAb1_2 0.11 nM huMAb1_3 0.12nM huMAb1_negA No binding detected huMAb1_negB 0.07 nM

Example 7.2.3: Cross Reactivity of huMAb1_1; huMAb1_2 and huMAb1_3 withCynomolgus Monkey LAMP1

HuMAb1_1, huMAb1_2 and huMAb1_3 antibodies were assessed by flowcytometry for their ability to bind to human LAMP1 or cynomolgus LAMP1proteins expressed respectively at the surface of HCT116 or HEK293stable clones. HCT116 stable clone was obtained as described in example4.7. HEK293 stable clone was obtained according to the protocoldescribed in example 4.7. EC₅₀s, estimated using BIOST@T-SPEED software,are listed in Table 28.

TABLE 28 Apparent affinity of huMAb1_1; huMAb1_2 and huMAb1_3 to humanLAMP1 or cynomolgus monkey LAMP1 EC₅₀ (nM) HCT116 HEK293 huLAMP1cynoLAMP1 Ratio clone 8 clone 44 of EC₅₀s huMAb1_1 15.0 30.1 2.0huMAb1_2 6.6 13.3 2.0 huMAb1_3 10.3 17.7 1.7

The result show, that huMAb1_1 binds to LAMP1 of human and cynomolgusorigin with similar affinity with a ratio of EC_(50s) of 2. HuMAb1_1cross-reacts with cynomolgus LAMP1. HuMAb1_2 binds to LAMP1 of human andcynomolgus origin with similar affinity with a ratio of EC_(50s) of2.01. HuMAb1_2 cross-reacts with cynomolgus LAMP1. HuMAb1_3 binds toLAMP1 of human and cynomolgus origin with similar affinity with a ratioof EC_(50s) of 1.71. HuMAb1_3 cross-reacts with cynomolgus LAMP1.

Example 7.3: Identification of the Epitope Binding Site of huMAb1_1 byCrystallography Example 73.1: Obtention of Fab1/LAMP1 Complex

Expression and Purification of Fab from huMAb1_1

Recombinant Fab from huMAb1_1 (Fab1) was obtained from transientlytransfected HEK293 cells, using two plasmids encoding the light chainLC1 or the C-terminal His-tagged heavy chain derived from HC1 with SEQID NO: 68 and 69, respectively. After cell clarification, growthsupernatant was applied on an immobilized-metal affinity resin (IMAC).After elution from the resin, the fractions containing highly pure Fab1were pooled & extensively dialysed against PBS. Fab1 solution was storedat 4° C.

Expression and Purification of Human Non-Glycosylated LAMP1-29-195

In order to obtain a non-glycosylated LAMP1 domain, bacterial expressionsystem was used. A thioredoxin fusion protein was designed forexpressing the domain L1-L2 of human LAMP1 protein with SEQ ID NO:70(TrxA-His-Thr-LAMP1-29-195 where Thr means thrombin cleavage site).The fusion protein was expressed using a T7 promoter, in a trxB-gordeficient E. coli strain. High cell density culture of the recombinantstrain was performed in a proprietary chemically defined medium, inbioreactor. From cell paste, the fusion protein was extracted by Frenchpress lysis, the cell lysate was clarified by ultracentrifugation andthe clarified supernatant was applied on an IMAC column. After elutionfrom the resin, the fractions containing the recombinant protein werepooled and the thrombin protease (Sigma-Aldrich) was used for cleavingoff the TrxA-His, one hour at room temperature. The solution was thenapplied on a Benzamidine Sepharose column (GE Healthcare) and on an IMACcolumn, for removing the thrombin and the free TrxA-His fusion partner,respectively. Purified untagged LAMP1-29-195 domain was stored at 4° C.till complex preparation. The sequence of untagged LAMP1-29-195 domainis referenced under SEQ ID NO: 71.

Preparation and Purification of the Complexes

Recombinant Fab (Fab1) and antigen (untagged LAMP1-29-195 domain) weremixed at a 1.5:1 molar ratio, incubated 30 min at room temperature & thecomplexes were further purified by preparative size exclusion on aSuperdex 200 PG column (GE Healthcare), equilibrated with PBS. Thefractions containing highly pure complex were pooled & stored at 4° C.till crystallography assays.

Example 73.2: Structure Determination of Fab1/LAMP1 Crystallization andData Collection

The complex was concentrated to 12 mg/mL in PBS10 mM pH7.Crystallization was done using the sitting drop method. It crystallizedin 20% PEG3350, 200 mM NaF (cond1) and 20% PEG3350, 200 mM DL-Malic acidpH7 (cond2). 25% ethylene glycol was included as cryoprotectant priorfreezing.

Datasets were collected from both crystals at beamline ID29 ESRF.Crystals diffracted in the same spacegroup C2 at 2.37 Å (cond1) or 2.51Å (cond2).

Structure Solution

A model of the constant domain of the Fab1 was obtained using the PDBstructure referenced under 4JG0. A model of the variable domain wasconstructed in Maestro (Schroedinger).

Molecular replacement was carried out using Phaser (Coy et al, J. Appl.Cryst. (2007) 40, 658-674) of the CCP4 suite (Winn et al, Acta Cryst D67(2011), 235-242) in both datasets but was successful only in cond2: aninitial refinement run confirmed the presence of two Fabs in theasymmetric unit. Additional density was visible above the variabledomains but too partial to place the antigen.

A second molecular replacement was carried out in cond1, using theresults from the previous run in cond2. This time, clear density couldbe visible above one of the variable domain, allowing the manualconstruction of a Lamp1 molecule. The second Lamp1 molecule was thenconstructed using the non-crystallographic symmetry between the twocomplexes.

The structure was refined using Buster (Buster-TNT 2.11.5, GlobalPhasing Ltd) at 2.37 Å to a Rfree of 26.1% and Rfactor 22.6%

Results

There are two Fab1/LAMP1 complexes in the asymmetric unit, withsignificantly different overall temperature factors. Interactionsbetween the two proteins are identical in both complexes; inconsequence, the most stable complex was taken as reference foranalysis.

The first luminal domain of LAMP1 corresponding to amino acids Ala29 toArg195 of SEQ ID NO: 24 interacts mostly with the heavy chain of Fab1.In FIG. 13 are indicated the residues of Fab1 which are part of theparatope (ie residues with atoms within 4A of the antigen atoms). Theamino acids that are part of the paratope are localized in the lightchain, more exactly in the CDR1-L with Asp30, Arg31 and Tyr32 of SEQ IDNO: 68 and CDR2-L with Asp50 of SEQ ID NO: 68. They are furtherlocalized in the heavy chain, more precisely in CDR1-H (Ile28, Asn31,Tyr32, Asn33), CDR2-H (Tyr52, Asn55, Asp57, Pro59), in FR3 loop (Thr74,Ser75, Ser77) and CDR3-H (Asn100, Trp101, Asp102, Phe105) SEQ ID NO: 69.

The epitope of LAMP1 for Fab1, determined as residues with atoms within4A of the Fab1 atoms, is indicated in bold in the sequence below anddisplayed in FIG. 14:

(SEQ ID NO 80) GSHMAMFMVKNGNGTACIMANFSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTLHDATIQAYLSNSSFSRGETRCEQDR

Said epitope identified by crystallography consist of the amino adisAsn35, Cys80, Gly 81, Glu83, Asn84, Arg106, Asn107, Ala108, Ile149,Asp150, Lys151, Tyr 178, Ser180, Asn181, Arg186 and Gly187 of SEQ IDNO:24.

As visible in the alignment displayed in FIG. 1, there is only oneresidue difference in the epitope region between Macacus fascicularisand Homo sapiens: at Position 187 of SEQ ID NO: 24 with Gly187Glu. Amodel of this mutation was built and minimized: the results show thatbinding to Fab1 remains possible provided side-chain movement of lightchain Tyr32 and slight movement of LAMP1 Lys151 as indicated in FIG. 15.

Based on the crystallographic structure that was herein obtained, it ispossible to suggest regions for affinity maturation. Two hot spots maybe the amino acid Ile28 in the heavy chain sequence of SEQ ID NO: 53 andthe amino acid Asn55 in the heavy chain sequence of SEQ ID NO: 53.Replacing the hydrophobic residue Ile 28 by a Glutamine might lead tointeraction with nearby Gly81 and Asn35 of LAMP1 (SEQ ID: 24) and shouldimprove binding between the two proteins as shown in FIG. 16. MutatingAsn55 to an Arginine would add interactions to Asn87, Arg106 and Thr107of LAMP1 (SEQ ID: 24) as shown in FIG. 17.

Example 8: Production and Characterization of ADC Example 8.1:Production and Characterization of ADC with a Maytansinoid DARCalculation:

A conjugate comprises generally from 1 to 10 molecule(s) of themaytansinoid attached covalently to the antibody (so called,“drug-to-antibody ratio” or “DAR”). This number can vary with the natureof the antibody and of the maytansinoid used along with the experimentalconditions used for the conjugation (like the ratiomaytansinoid/antibody, the reaction time, the nature of the solvent andof the cosolvent if any). Thus the contact between the antibody and themaytansinoid leads to a mixture comprising: several conjugates differingfrom one another by different drug-to-antibody ratios; optionally thenaked antibody; optionally aggregates. The DAR that is determined isthus a mean value.

The method used herein to determine the DAR consists in measuringspectrophotometrically the ratio of the absorbance at 252 nm and 280 nmof a solution of the substantially purified conjugate. In particular,said DAR can be determined spectrophotometrically using the measuredextinction coefficients at respectively 280 and 252 nm for the antibody(ε_(A280) and ε_(A252)) and for the maytansinoid (ε_(D280)=5,180 M⁻¹cm⁻¹and ε_(D252)=26,159 M⁻¹cm⁻¹). The method of calculation is derived fromAntony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies andProtocols, vol 525, 445, Springer Science and is described in moredetails below:

The absorbances for the conjugate at 252 nm (A252) and at 280 nm (A280)are measured on a spectrophotometer apparatus to calculate the DAR. Theabsorbances can be expressed as follows:

A ₂₅₂=(C _(D)×ε_(D252))+(C _(A)×ε_(A252))

A ₂₈₀=(C _(D)×ε_(D280))+(C _(A)×ε_(A280))

wherein:

-   -   C_(D) and C_(A) are respectively the concentrations in the        solution of the maytansinoid and of the antibody    -   ε_(D252) and ε_(D280) are respectively the molar extinction        coefficients of the maytansinoid at 252 nm and 280 nm    -   ε_(A252) and ε_(A280) are respectively the molar extinction        coefficients of the antibody at 252 nm and 280 nm.

Resolution of these two equations with two unknowns leads to thefollowing equations:

C _(D)=[(ε_(A280) ×A ₂₅₂)−(ε_(A252) ×A₂₈₀)]/[(ε_(D252)×ε_(A280))−(ε_(A252)×ε_(D280))]

C _(A) =[A ₂₈₀−(C _(D)×ε_(D280))]/ε_(A280)

The average DAR is then calculated from the ratio of the drugconcentration to that of the antibody:

DAR=C _(D) /C _(A)

Deglycosylation and High Resolution Mass Spectrometry of Conjugates(HRMS)

Deglycosylation is a technique of enzymatic digestion by means ofglycosidase. The deglycosylation is made from 50 μl of conjugated+10 μlof N-glycosidase-F (PN Gase F) enzyme (100 units of freeze-driedenzyme/100 μl of water). The medium is vortexed and maintained one nightat 37° C. The deglycosylated sample is then ready to be analyzed inHRMS. Mass spectra were obtained on a Waters XEVO QT of system inelectrospray positive mode (ES+). Chromatographic conditions are thefollowing: column: UPLC BEH300 C4 1.7 μm 2.1×150 mm; solvents: A:H₂O+0.1% formic acid: B; CH₃CN+0.1% formic acid; column temperature: 70°C.; flow rate 0.5 mL/min; gradient elution (10 min): 20% B for 2 min 50sec; from 20 to 80% of B in 2 min 5 sec; 8 min 50 sec: 80% B; 8 min 55sec: 20% B; 10 min: 20% B.

Buffers Content

-   -   Buffer A (pH 6.5): NaCl (50 mM), Potassium Phosphate buffer (50        mM), EDTA (2 mM)    -   Buffer HGS (pH 5.5): histidine (10 mM), glycine (130 mM),        sucrose 5% (w/v), HCl (8 mM)    -   PBS (pH 7.4): KH₂PO₄ (1.03 mM), NaCl (155.17 mM), Na₂HPO₄-7H₂O        (2.97 mM)

Abbreviations Used

-   -   DAR: Drug Antibody Ratio; DMA: dimethylacetamide; HEPES:        4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid; HRMS: High        Resolution Mass Spectroscopy; Nitro-SPDB: butanoic acid,        4-[(5-nitro-2-pyridinyl)dithio]-, 2,5-dioxo-1-pyrrolidinyl ester        (could be prepared as described in WO2004016801 patent); RT:        room temperature.

Example 8.1.1: Antibody Drug Conjugate (ADC) (Chimer chMAb 1)

The naked chimer of MAb1 was prepared. It is a chimeric mAb (chMAb1)derived from the murine clone MAb1 with a human IgG1, Ck isotype havinga heavy chain of sequence SEQ ID NO: 17 and a light chain of sequenceSEQ ID NO: 18.

In this example, a cleavable conjugate (indifferently calledDM4-SPDB-chMAb1 or chMAb1-SPDB-DM4) was obtained from the naked chMAb1,as described in example 7, and from DM4 with LC and HC sequences of thechimeric mAb corresponding to SEQ ID NO: 17 and 18.

DM4 has the following chemical nameN^(2′)-deacetyl-N^(2′)-(4-mercapto-4-methyl-1-oxopentyl) maytansine.

For chMAb1, ε_(A280)=223,400 M⁻¹cm⁻¹ and ε_(A252)=80,240 M⁻¹cm⁻¹

Preparation and Analytical Data of the Cleavable Chimer chMAb1-SPDB-DM4Conjugate

To 12.1 mL of a solution of chMAb1 antibody at a concentration of 8.23mg/mL in buffer A is added under magnetic stirring 465 μL of HEPES 1N, 1mL DMA and a 5.9-fold molar excess of a 15 mM solution of nitro-SPDB inDMA. After 1 h30 at RT, the reaction mixture is diluted with 17.3 mL ofPBS buffer and 1.64 mL DMA, prior to addition of a 15 mM solution of DM4in DMA.

The reaction mixture is stirred at RT for 2 h40 and then purified by TFFon Sius-LSn Prostream 30 kD cassette. The sample is diafiltrated against600 mL of HGS buffer, concentrated, collected and filtered over Millex®0.22 μM PVDF filter to yield product (11 mL).

The final conjugate is assayed spectrophotometrically; an average DAR of4.5 DM4 per molecule of antibody (5.7 mg/mL) was determined. HRMS data:see FIG. 7.

Example 8.1.2: Antibody Drug Conjugate (Humanized huMAb1_3)

The naked humanized huMAb1_3 was prepared as described in example 7.2.2.It is a humanized mAb derived from the murine clone MAb1 with a humanIgG1 and Ck isotype, Ck isotype having a heavy chain (VH3-hulgG1)sequence of SEQ ID NO: 64 and a light chain (VL3-huCk) sequence SEQ IDNO: 63.

-   Extinction coefficients mentioned herein were measured at    respectively 280 and 252 nm for the antibody ε_(A280)=223,400    M⁻¹cm⁻¹ and ε_(A252)=79,413

Preparation of DM4-SPDB-huMAb1_3 Conjugate

To 3.6 mL of a solution of huMAb1_3 antibody at a concentration of 5.18mg/mL in DPBS is added under magnetic stirring, 0.316 mL DMA and a5.2-fold molar excess of a 15 mM solution of nitro-SPDB in DMA. After 2h15 at RT 0.3 equivalent of a 15 mM solution of nitro-SPDB in DMA isadded. After 1 h45 the reaction mixture is diluted with 1.99 mL of PBSbuffer and 0.187 mL DMA, prior to addition of a 15 mM solution of DM4 inDMA.

The reaction mixture is stirred at RT for 2 h and then purified by gelfiltration (HiPrep 26/10 desalting, Sephadex G25, GE Healthcare)previously equilibrated with buffer HGS pH=5.5. The collected sample isfiltered over Millex® 0.22 μM PVDF filter to yield product (9.4 mL).This solution is injected on a Chromasorb® Millipore 0.08 mL device(CHRFA1PD09), The collected sample is concentrated over Amicon Ultra-1510KD, Millipore and filtered over Millex® 0.22 μM PVDF filter to yieldproduct (4.3 mL). The final conjugate is assayed spectrophotometrically;an average DAR of 3.5 DM4 per molecule of antibody (1.9 mg/mL) wasdetermined. HRMS data: see FIG. 18.

Example 8.1.3: Antibody Drug Conjugate (humanized huMAb1_1)

The naked humanized huMAb1_1 was prepared as described in example 7.2.2.It is a humanized mAb derived from the murine clone MAb1 with a humanIgG1 and Ck isotype, Ck isotype having a heavy chain (VH1-hulgG1)sequence of SEQ ID NO: 60 and a light chain sequence (VL1-huCk) of SEQID NO: 59 Extinction coefficients are measured at respectively 280 and252 nm for the antibody

ε_(A280)=223,400 M⁻¹cm⁻¹ and ε_(A252)=80,041 M⁻¹ cm⁻¹

Preparation of DM4-SPDB-huMAb1_1 Conjugate

To 3.6 mL of a solution of huMAb1_1 antibody at a concentration of 4.81mg/mL in DPBS is added under magnetic stirring, 0.321 mL DMA and a5.0-fold molar excess of a 15 mM solution of nitro-SPDB in DMA. After 2h at RT 0.3 equivalent of a 15 mM solution of nitro-SPDB in DMA isadded. After 1 h30 the reaction mixture is diluted with 1.59 mL of PBSbuffer and 0.147 mL DMA, prior to addition of a 15 mM solution of DM4 inDMA.

The reaction mixture is stirred at RT for 1 h30 and then purified by gelfiltration (HiPrep 26/10 desalting, Sephadex G25, GE Healthcare)previously equilibrated with buffer HGS pH=5.5. The collected sample isfiltered over Millex® 0.22 μM PVDF filter to yield 9.4 mL of solution.This solution is injected on a Chromasorb® Millipore 0.08 mL device(CHRFA1PD09), The collected sample is concentrated over Amicon Ultra-1510KD, Millipore and filtered over Millex® 0.22 μM PVDF filter to yieldproduct (4.9 mL). The final conjugate is assayed spectrophotometrically;an average DAR of 3.4 DM4 per molecule of antibody (1.45 mg/mL) wasdetermined. HRMS data: see FIG. 19.

Example 8.1.4: Antibody Drug Conjugate (Humanized huMAb1_2)

The naked humanized huMAb1_2 was prepared as described in example B7. Itis a humanized mAb derived from the murine clone MAb1 with a human IgG1and Ck isotype, Ck isotype having a heavy chain sequence (VH2-hulgG1) ofSEQ ID NO: 62 and a light chain sequence (VL2-huCk) SEQ ID NO: 61.

Extinction coefficients are measured at respectively 280 and 252 nm forthe antibody ε_(A250)=223,400 M⁻¹cm⁻¹ and ε_(A252)=79,474

Preparation of DM4-SPDB-huMAb1_2 Conjugate

To 3.6 mL of a solution of huMAb1_2 antibody at a concentration of 5.06mg/mL in b DPBS is added under magnetic stirring, 0.317 mL DMA and a5.2-fold molar excess of a 15 mM solution of nitro-SPDB in DMA. After 2h the reaction mixture is diluted with 1.89 mL of PBS buffer and 0.179mL DMA, prior to addition of a 15 mM solution of DM4 in DMA.

The reaction mixture is stirred at RT for 1 h30 and then purified by gelfiltration (HiPrep 26/10 desalting, Sephadex G25, GE Healthcare)previously equilibrated with buffer HGS pH=5.5. The collected sample isfiltered over Millex® 0.22 μM PVDF filter to yield product (9.7 mL).

The final conjugate is assayed spectrophotometrically; an average DAR of4.05 DM4 per molecule of antibody (1.36 mg/mL) was determined. HRMSdata: see FIG. 20.

Example 8.1.5: Antibody Drug Conjugate (Chimer chMAb2can)

The naked chimer chMAb2can was prepared as described in example 7. It isa chimer mAb derived from the murine clone MAb2 with a human IgG1 and Ckisotype. Ck isotype having a heavy chain sequence of SEQ ID NO: 21 and alight chain of sequence SEQ ID NO: 22.

Extinction coefficients at respectively 280 and 252 nm for the antibodyε_(A280)=223,400 M⁻¹cm⁻¹ and ε_(A252)=74,417 M⁻¹cm⁻¹

Preparation of DM4-SPDB-chMAb2can Conjugate

To 9.2 mL of a solution of chMAb2can antibody at a concentration of 5.21mg/mL in DPBS is added under magnetic stirring, 0.813 mL DMA and a5.0-fold molar excess of a 15 mM solution of nitro-SPDB in DMA. After 2h at RT 0.2 equivalent of a 15 mM solution of nitro-SPDB in DMA isadded. After 1 h30 the reaction mixture is diluted with 5.17 mL of PBSbuffer and 0.497 mL DMA, prior to addition of a 15 mM solution of DM4 inDMA.

The reaction mixture is stirred at RT for 1 h30 and then purified by gelfiltration (HiPrep 26/10 desalting, Sephadex G25, GE Healthcare)previously equilibrated with buffer HGS pH=5.5. The collected sample isfiltered over Millex® 0.22 μM PVDF filter to yield product (23 mL).

The final conjugate is assayed spectrophotometrically; an average DAR of3.7 DM4 per molecule of antibody (1.58 mg/mL) was determined. HRMS data:see FIG. 21.

Example 8.1.6: Antibody Drug Conjugate (chimer chMAb3 VLR24-R93)

The naked chimer chMAb3_VLR24-R93 was prepared as described in example7. It is a chimer mAb derived from the murine clone MAb3 with a humanIgG1 and Ck isotype, Ck isotype having a heavy chain sequence of SEQ IDNO: 49 and a light chain of sequence SEQ ID NO: 81.

Extinction coefficients are measured at respectively 280 and 252 nm forthe antibody ε_(A280)=234539 M⁻¹cm⁻¹ and ε_(A252)=85303 M⁻¹cm⁻¹.

Preparation of DM4-SPDB-chMAb3 VLR24-R93 conjugate

To 7.3 mL of a solution of chMAb3_VLR24-R93 antibody at a concentrationof 6.85 mg/mL in DPBS were added under magnetic stirring, 7.7 mL of PBS,1.5 mL DMA and a 5.0-fold molar excess of a 10 mM solution of nitro-SPDBin DMA. After 3 h30, 155 μL of a solution of L-DM4 (15.1 mM in DMA) wereadded to the reaction mixture. The reaction mixture was stirred at RTfor 1 h30 and then purified by gel filtration (HiPrep 26/10 desalting,Sephadex G25, GE Healthcare) previously equilibrated with buffer HGSpH=5.5. The collected sample was filtered over Millex® 0.22 μM PVDFfilter to yield product (23 mL). The final conjugate was assayedspectrophotometrically; an average DAR of 3.7 DM4 per molecule ofantibody (2.0 mg/mL) was determined. HRMS data: see FIG. 22.

Example 8.1.7: Cross Reactivity of DM4-SPDB-chMAb1, DM4-SPDB-chMAb2, andDM4-SPDB-chMAb3, to Human LAMP1/Cyno LAMP1

DM4-SPDB-chMAb1, DM4-SPDB-chMAb2 and DM4-SPDB-chMAb3 were assessed byflow cytometry for their ability to bind to human LAMP1 or cynomolgusLAMP1 proteins expressed respectively at the surface of HCT116 or HEK293stable clones. HCT116 stable clone and HEK293 stable clone were obtainedas described in the protocol in example 4.7 EC_(50s), estimated usingBIOST@T-SPEED software, are listed in Table 29.

TABLE 29 Apparent affinity of DM4-SPDB-chMAb1, DM4-SPDB-chMAb2,DM4-SPDB-chMAb3 to human LAMP1 or cynomolgus monkey LAMP1 EC₅₀ (nM)HCT116 HEK293 huLAMP1 cynoLAMP1 Ratio clone 8 clone 44 of EC_(50s)DM4-SPDB-chMAb1 6.6 6.6 1.0 DM4-SPDB-chMAb2 5.5 12.8 2.3 DM4-SPDB-chMAb39.1 6.1 0.7

DM4-SPDB-chMAb1 binds to LAMP1 of human and cynomolgus origin withsimilar affinity. EC_(50s) ratio was 1 and therefore DM4-SPDB-chMAb1cross-reacted with cynomolgus LAMP1. DM4-SPDB-chMAb2 binds to LAMP1 ofhuman and cynomolgus origin with similar affinity. EC_(50s) ratio was2.3 and therefore DM4-SPDB-chMAb2 cross-reacted with cynomolgus LAMP1.DM4-SPDB-chMAb3 binds to LAMP1 of human and cynomolgus origin withsimilar affinity. EC_(50s) ratio was 0.7 and therefore DM4-SPDB-chMAb2cross-reacted with cynomolgus LAMP1.

Example 8.1.8: Cross Reactivity of DM4-SPDB-huMAb1_1 to Human LAMP1/CynoLAMP1

DM4-SPDB-huMAb1_1 was assessed by flow cytometry for its ability to bindto human LAMP1 or cynomolgus LAMP1 proteins expressed respectively atthe surface of HCT116 or HEK293 stable clones, both obtained asdescribed in the protocol of example 4.7. EC₅₀s, estimated usingBIOST@T-SPEED software, are listed in Table 30.

TABLE 30 Apparent affinity of DM4-SPDB-huMAb1_1 to human LAMP1 orcynomolgus monkey LAMP1 EC₅₀ (nM) HCT116 HEK293 huLAMP1 cynoLAMP1 Ratioclone 8 clone 44 of EC₅₀s DM4-SPDBhuMAb1_1 12.65 33.50 2.65

DM4-SPDB-huMAb1_1 binds to LAMP1 of human and cynomolgus origin withsimilar affinity with a ratio of EC_(50s) of 2.65. Therefore,DM4-SPDBhuMAb1_1 cross-reacts with cynomolgus LAMP1.

Example 8.2: Production and Characterization of ADC with a TomaymycineDimer DAR Calculation:

DAR calculation is determined similarly than for maytansinoid ADC, usingthe measured extinction coefficients at respectively 280 and 322 nm forthe antibody (ε_(A280)=223,400 M⁻¹cm⁻¹ and ε_(A322) ⁼⁰ M⁻¹cm⁻¹) and forthe tomaymycine dimer (ε_(D280)=4,436 M⁻¹cm⁻¹ and ε_(D322)=7,843M⁻¹cm⁻¹).

Preparation of a huMAb1_1 conjugate modified with SNPP (N-succinimidyl4-(5-nitro-2-pyridyldithio)pentanoate) with(2E,2′E,11aS,11a'S)-8,8′-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2, 3-dihydro-1Hbenzo[e]pyrrolo[1,2-a][1, 4] diazepin-5(11aH)-one)

To 56 mg of huMAb1_1 in 2.8 ml of buffer A are added 0.48 mg of SNPP(N-succinimidyl 4-(5-nitro-2-pyridyldithio)pentanoate) dissolved in 67μL of DMA under stirring. After 3 hours at room temperature, thesolution of modified antibody is fractionated into two and purified bygel filtration on two Sephadex G25 columns (PD-10 GE column)pre-equilibrated in an aqueous buffer with a concentration of 0.05 MN-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES), 0.05 MNaCl and 2 mM ethylenediaminetetraacetic acid (EDTA) of pH=8. Aftermixing and homogenizing the two filtrates thus obtained, the modifiedantibody is assayed by spectrophotometry using the extinctioncoefficients of nitropyridinethiol (ε_(280 nm)=3344 M⁻¹ cm⁻¹ andε_(325 nm)=10964 M⁻¹cm⁻¹): an average of 3.32 dithio-nitropyridinegroups per antibody molecule was determined at a concentration of 6.28mg/mL.

To 9.4 mg of modified antibody above in 1.5 ml of an aqueous buffer witha concentration of 0.05 MN-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES), 0.05 MNaCl and 2 mM ethylenediaminetetraacetic acid (EDTA) of pH=8 are added0.56 mL of DMA and 1.12 mg of(2E,2′E,11aS,11a'S)-8,8′-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2,3-dihydro-1Hbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one) dissolved in 0.06 mL ofdimethylacetamide (DMA) under stirring. After 17 at 30° C., 0.01N HCl isadded until pH=6.6 and the resulting mixture is purified on a CHT 80(type II) column (20 mm×8 mm I.D.) initially equilibrated with 2 mL of a200 mM potassium phosphate buffer of pH 6.5 followed by 4 mL of a 10 mMpotassium phosphate buffer of pH 6.5. After injection and washing with 5mL of the last 10 mM phosphate buffer, elution is realized with 6 mL ofthe previous 200 mM phosphate buffer. 2.5 mL of the resulting batch isthen filtered on a Sephadex G25 column (PD-10 GE column)pre-equilibrated in an aqueous buffer of pH=6.5 with a concentration of10 mM histidine, containing 10% sucrose.

The chemical structure for(2E,2′E,11aS,11a'S)-8,8′-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2,3-dihydro-1Hbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one) is as follows:

The conjugate obtained (3.5 mL) is assayed by spectrophotometry: anaverage of 2.85(2E,2′E,11aS,11a'S)-8,8′-(((4-(2-(2-(2-((2-mercapto-2-methylpropyl)(methyl)amino)ethoxy)ethoxy)ethoxy)pyridine-2,6-diyl)bis(methylene))bis(oxy))bis(2-ethylidene-7-methoxy-2,3-dihydro-1Hbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one) per antibody molecule wasdetermined at a concentration of 1.84 mg/mL. HRMS data: see FIG. 37.

Example 9: In Vitro Cytotoxicity Example 9.1: In Vitro Cytotoxicity ofDM4-SPDB-chMAb1

HCT116 cells were infected by a lentiviral vector allowing stableintegration of the LAMP1 CDS in genomic DNA of cells, as reported inexample 4.7 Individual clones with different densities of LAMP1 cellsurface localization were derived from pool of HCT116 infected cells.HCT116 clone 8 was used to compare EC50 of chMAb1 and DM4-SPDB-chMAb1.Cells were plated in 96-well plates at 200 000 per well and MAb1 orDM4-SPDB-chMAb1 was added in 2-fold serial dilution up to 12 dilutionsin assay diluant for 1 h at 4° C. and washed two times with PBS 1% BSA.100 μL/well of goat anti-human IgG conjugated with Alexa488 (Invitrogen;# A11013) was added for 1 h at 4° C. and washed two times with PBS 1%BSA. The antibody binding was evaluated after centrifugation andresuspension of cells in 100 ul fixing solution (paraformaldehyde at 4%in PBS). Samples were read using Galaxy® Flow Cytometry System (Partec).EC50 values were estimated using BIOST@T-SPEED software. chMAb1 andDM4-SPDB-chMAb1 bind with similar affinity and EC₅₀ of 6.0 and 6.6 nMrespectively.

Several HCT116 clones with different antigen densities were used toevaluate cytotoxicity of DM4-SPDB-chMAb1 by assessment of cell viabilityusing the Cell Titer-Glo kit (Promega).

HCT116-LAMP1 cells were plated in 96-well plates and allowed to adhereduring 4 hours in 37° C./5% CO₂ atmosphere. Different concentrations ofDM4-SPDB-chMAb1 were added to the seeded cells, in triplicate for eachconcentration. The cells were then incubated for 96 hours in the sameatmosphere. Cell Titer-Glo reagent was then added to the wells for 10min at room temperature and the luminescent signal was measured using aVictor plate reader (Perkin-Elmer). The half maximal inhibitoryconcentration (IC₅₀) is a measure of the effectiveness of a compound ininhibiting biological function. It is defined as the concentration ofthe antibody which led to cell killing with a response halfway betweenthe baseline and maximum after some specified exposure time. The IC₅₀values were estimated using BIOST@T-SPEED software. DM4-SPDB-chMAb1killed cells with an IC₅₀ of around 0.2 nM.

TABLE 31 Cytotoxicity of DM4-SPDB-chMAb1 on HCT116 cell lines expressingLAMP1 Cytotoxic activity IC₅₀ (nM) HCT116-LAMP1 Antigen density (mean ±SD; n = 3) Clone 5 451 000 0.2 ± 0.1 Clone 8 160 000 0.1 ± 0.1

Cytotoxic IC50 of DM4-SPDB-chMAb1 is sub-nM for clones of HCT116 withhigh expression of LAMP1.

Example 9.2: In Vitro Cytotoxicity of DM4-SPDB-huMAb1_1DM4-SPDB-huMAb1_2, DM4-SPDB-huMAb1_3

HCT116 clone 8, as described in example 9, was used to evaluatecytotoxicity of DM4-SPDB-huMAb1_1, DM4-SPDB-huMAb1_2 andDM4-SPDB-huMAb1_3. The same protocol as described in example 9 wasapplied.

The three constructs killed cells with an equivalent efficacy. IC₅₀ are0.10 nM, 0.07 nM and 0.12 nM for DM4-SPDB-huMAb1_1, DM4-SPDB-huMAb1_2and DM4-SPDB-huMAb1_3 respectively.

Example 9.3: In Vitro Cytotoxicity of DM4-SPDB-chMAb2 andDM4-SPDB-chMAb3

HCT116 clone 8, as described in example 9, was used to comparecytotoxicity of DM4-SPDB-chMAb2 and DM4-SPDB-chMAb3. Same protocol asdescribed in example 9 was applied. The two constructs killed cells withan equivalent efficacy. 10₅₀ are 0.07 nM and 0.06 nM for DM4-SPDB-chMAb2and DM4-SPDB-chMAb3 respectively.

Example 9.4: Evaluation of the Inhibition of Proliferation of the CellLines HCT116 and HT29 with ADC with Tomaymycine Dimer

HCT116 (respectively HT29) cells in their exponential growth phase aretrypsinized and resuspended in their culture medium (DMEMGibco#11960+10% SVF+2 mM glutamine). The cell suspension is seeded inCytostar 96-well plates (GE Healthcare Europe, #RPNQ0163) in wholeculture medium containing serum to a density of 3000 (respectively 5000)cells/well. After incubation for 4 hours, successive dilutions of theantibody-cytotoxic agent immunoconjugate are added to the wells atdecreasing concentrations from 10⁻¹ to 10⁻¹² M (in duplicate(respectively in quadruplate) for each concentration). The cells arecultured at 37° C. under an atmosphere containing 5% CO₂ in the presenceof the antibody-cytotoxic agent immunoconjugate for 3 days. On thefourth day, 10 μl of a solution 14C-thymidine (0.1 μCi/well, PerkinElmer #NEC56825000) are added to each well. The incorporation of14C-thymidine is measured 96 hours after the start of the experimentwith a Microbeta radioactivity counter (Perkin Elmer). The data areexpressed in the form of a percentage of survival by determining theratio between accounts obtained with the cells treated with theimmunoconjugate and that obtained with the cells of the control wells(treated with the culture medium alone).

The construct killed the HCT116 and HT29 cells with an equivalentefficacy. IC₅₀ are 56 pM for the HCT116 cells and 72 nM for the HT29cells.

Example 10: In Vivo Efficacy Example 10.1: In Vivo Efficacy ofDM4-SPDB-chMAb1

In this example, the cleavable DM4-SPDB-chMAb1 conjugate was shown tolead to in vivo efficacy.

Example 10.1.1: Evaluation of the Antitumor Activity of DM4-SPDB-chMAb1Against Primary Human Colon Adenocarcinoma CR-LRB-010P Materials andMethods

The conjugate DM4-SPDB-chMAb1 was evaluated at 3 doses againstmeasurable primary colon CR-LRB-010P tumors implanted subcutaneously infemale SCID mice. Control groups were left untreated. DM4-SPDB-chMAb1was administered at 10, 5 and 2.5 mg/kg by an intravenous (IV) bolusinjection, on day 17 post tumor implantation. Animals were weighed dailyand tumors were measured 2 times weekly by caliper. A dosage producing a20% weight loss or 15% weight loss for 3 consecutive days or 10% or moredrug deaths, was considered an excessively toxic dosage. Animal bodyweights included the tumor weights. Tumor volumes were estimated from 2dimensional tumor measurements and calculated according to the followingformula (Corbett, T. H. et al., 1977, Cancer 40: 2660-2680): Tumorvolume (mm³)=[Length (mm)×Width² (mm²)]/2. The primary efficacy endpoints are ratio of change in tumor volume changes from baseline betweentreated and control groups (ΔT/ΔC), percent median regression, partialregression (PR) and complete regression (CR).

Changes in tumor volume for each treated (T) and control (C) arecalculated for each tumor by substracting the tumor volume on the day offirst treatment from the tumor volume on the specified observation day.The median ΔC is calculated for the untreated control group and themedian ΔT is calculated for the treated group. Then the ratio ΔT/ΔC iscalculated and expressed as a percentage: ΔT/ΔC=(delta T/delta C)×100 asdescribed before

The dose is considered as therapeutically active when ΔT/ΔC is lowerthan 40% and very active when ΔT/ΔC is lower than 10%. If ΔT/ΔC is lowerthan 0, the dose is considered as highly active and the percentage ofregression is dated (Plowman J, Dykes D J, Hollingshead M,Simpson-Herren L and Alley M C. Human tumor xenograft models in NCI drugdevelopment. In: Feibig H H BA, editor. Basel: Karger.; 1999 p 101-125):

% tumor regression: is defined as the % of tumor volume decrease in thetreated group at a specified observation day compared to its volume onthe first day of first treatment.

At a specific time point and for each animal, % regression iscalculated. The median % regression is then calculated for the group.

${\% \mspace{14mu} {regression}\mspace{14mu} \left( {{at}\mspace{11mu} t} \right)} = {\frac{{volume}_{t\; 0} - {volume}_{t}}{{volume}_{t\; 0}} \times 100}$

Partial regression (PR): Regressions are defined as partial if the tumorvolume decreases to 50% of the tumor volume at the start of treatment.

Complete regression (CR): Complete regression is achieved when tumorvolume=0 mm³ (CR is considered when tumor volume cannot be recorded).

Results:

The results are presented on FIG. 5 and Table 32 (below).DM4-SPDB-chMAb1 given at 10.0, 5.0 and 2.5 mg/kg was well tolerated witha maximal body weight loss of 3.7% at 10 mg/kg. After a singleadministration at 10.0 and 5.0 mg/kg DM4-SPDB-chMAb1 was highly activeand statistically significant (p<0.0001 for each dose), as compared tocontrol, producing a ΔT/ΔC<0 and regressions of the initial tumor volumeof 75% and 7%, respectively with 5/6 PR and 2/6 CR at 10 mg/kg and 1/6PR at 5 mg/kg. The dosage below 2.5 mg/kg was active with ΔT/ΔC=31% andno regressions.

TABLE 32 Evaluation of the anti-tumor activity of DM4-SPDB-chMAb1against primary human colon adenocarcinoma CR-LRB-010P in SCID femalemice Average body Route/ Dosage Drug weight change Median % Dosage in inmg/kg death in % per Median of Tumor free Biostatistic mL/kg per (totalSchedule (Day of mouse at nadir ΔT/Δc regression Regressions survivorsat p value Agent¹ injection dose) in days deatch) (day of nadir) in %(day) (day) Partial Complete day 55 (day) ² DM4- IV (10 10.0 (10.0) 170/6 −3.7 (18) −11 (31)  75 (31) 5/6 2/6 0/6 P < 0.0001 SPDB- mL/Kg) (31)chMAb1 DM4- IV (5 5.0 (5.0) 0/6 −3.2 (18) −2 (27)  7 (27) 1/6 0/6 0/6 P< 0.0001 SPDB- mL/Kg) (27) chMAb1 DM4- IV (2.5 2.5 (2.5) 0/6 +0.1 (18)31 (31) 0/6 0/6 0/6 P = 0.0001 SPDB- mL/Kg) (31) chMAb1 Controls  0/10−2.7 (18)  0/10  0/10  0/10 ¹Drug formulation: buffer HGS pH 5.5 (10 mMhistidine, 130 mM glycine, 5% (w/v) sucrose, 0.01% Tween 80) ² p-value:Dunnett's test versus control following a two-way Anova with repeatedmeasures performed separately for each compounds on ranks of changesfrom baseline. A probability less than 5% (p < 0.05) was considered assignificant. NS = non significant. Tumor doubling time = 4.3 days. Tumorsize at start of therapy was 96-216 mm³, with a median tumor burden pergroup of 126-138 mm³.

Example 10.1.2: Evaluation of the Antitumor Activity of DM4-SPDB-chMAb1Against Primary Human Lung Tumor LUN-NIC-0014 Materials and Methods

DM4-SPDB-chMAb1 was evaluated at 3 doses against measurable primary lungtumor LUN-NIC-0014 implanted s.c in female SCID mice. Control groupswere left untreated. DM4-SPDB-chMAb1 was administered at 10.0, 5.0 and2.5 mg/kg by an intravenous (IV) bolus injection, on day 26 post tumorimplantation. Animals were weighed daily and tumors were measured 2times weekly by caliper. A dosage producing a 20% weight loss or 15%weight loss for 3 consecutive days or 10% or more drug deaths, wasconsidered an excessively toxic dosage. Animal body weights included thetumor weights.

Toxicity and efficacy evaluation were performed as reported in example10-1.

Results

DM4-SPDB-chMAb1 given at 10.0, 5.0 and 2.5 mg/kg was well tolerated witha maximal body weight loss of 3.3% at 2.5 mg/kg. After a singleadministration at 10.0 and 5.0 mg/kg DM4-SPDB-chMAb1 was highly activeas compared to control, producing a ΔT/ΔC<0 and regressions of theinitial tumor volume of 81% and 65%, respectively, and with 6/6 CR at 10mg/kg and 5/6 PR at 5 mg/kg. The dosage below 2.5 mg/kg was active withΔT/ΔC=33% and no regressions. (Table 33, FIG. 6).

TABLE 33 Evaluation of the antitumor activity of DM4-SPDB-chMAb1 againstprimary human lung tumor LUN-NIC-0014 in SCID female mice Average bodyMedian Route/Dosage Dosage in weight change ΔT/Δc Median % of in mL/kgper mg/kg per in % (on day in % (on regression Regressions Agent¹injection injection of trough) day 42) (on day 42) Partial CompleteDM4-SPDB- 10 Single dose −2.52 (28) <0 81 6/6 6/6 chMAb1 mL/kg IV day 26DM4-SPDB- 5 Single dose −1.34 (28) <0 65 5/6 0/6 chMAb1 mL/kg IV day 26DM4-SPDB- 2.5 Single dose −3.32 (28) 33 0/6 0/6 chMAb1 mL/kg IV day 26Control −2.83 (27) 0/6 0/6 Drug formulation: HGS buffer at pH 5.5 (10 mMhistidine, 130 mM glycine, 5% (w/v) sucrose, 0.01% Tween 80) Tumordoubling time = 7.5 days. Tumor size at start of therapy was 99-230 mm³,with amedian tumor burden per group of 148-162 mm³.

Example 10.2: In Vivo Efficacy of DM4-SPDB-huMAb1_1 Example 10.2.1:Evaluation of the Anti-Tumor Activity of DM4-SPDB-huMAb1_1 AgainstPrimary Human Colon Adenocarcinoma CR-LRB-010P Material and Method

DM4-SPDB-huMAb1_1 was evaluated at 2 doses against measurable primarycolon CR-LRB-010P tumors implanted s.c in female SCID mice. Controlgroups were left untreated. DM4-SPDB-huMAb1_1 was administered at 5 and2.5 mg/kg by an intravenous (IV) bolus injection, on day 19 post tumorimplantation. Animals were weighed daily and tumors were measured 2times weekly by caliper.

Toxicity and efficacy evaluation were performed as reported in example10.1. Results

DM4-SPDB-huMAb1_1 given at 5.0 and 2.5 mg/kg was well tolerated with amaximal body weight loss of 4.4% at 2.5 mg/kg. After a singleadministration at 5.0 mg/kg DM4-SPDB-huMAb1_1 was active andstatistically significant (p<0.0001), as compared to control, producinga ΔT/ΔC=0 without regressions. The dosage below 2.5 mg/kg yielded aΔT/ΔC=47%. (Table 34, FIG. 23)

TABLE 34 Evaluation of the anti-tumor activity of DM4-SPDB-huMAb1_1against primary human colon adenocarcinoma CR-LRB-010P in SCID femalemice. Dosage in Average body Route/ mg/kg per Drug weight change MedianMedian % Dosage in injection death in % per ΔT/ΔC of mL/kg per (totalSchedule (Day of mouse at nadir in % regression Regressions BiostatisticAgent injection dose) in days death) (day of nadir) day 25 on dayPartial Complete p value (25) DM4-SPDB- IV 10 5 (5) 19 0/6 −0.9 (21) 0 —0/6 0/6 p < 0.0001 huMAb1_1 mL/kg 2.5 (2.5) 0/6 −4.4 (45) 47 — 0/6 0/6 p= 0.2258 Control  0/10 −3.5 (37) — — 0/6 0/6 — Tumor doubling time = 6.0days. Tumor size at start of therapy was 88-277 mm³, with a median tumorburden per group of 138-157 mm³. Mice average weight range = 18.10-22.40g dosages were adjusted to the individual body weights. Drugformulation: His-Gly-Sucrose buffer pH = 5.5; Statistical analysis:Dunnett's test versus control following a two-way Anova with repeatedmeasures performed separately for each compounds on ranks of changesfrom baseline. A probability less than 5% (p < 0.05) was considered assignificant.

Example 10.2.2: Evaluation of the Anti-Tumor Activity ofDM4-SPDB-huMAb1_1 Against Primary Invasive Ductal Carcinoma BRE-IGR-0159Material and Method

DM4-SPDB-huMAb1_1 was evaluated at four doses against measurable primaryinvasive ductal carcinoma BRE-IGR-0159 tumors implanted s.c in femaleSCID mice. Control groups were left untreated. DM4-SPDB-huMAb1_1 wasadministered at 5, 2.5, 1.25 and 0.62 mg/kg by an intravenous (IV) bolusinjection, on day 17 post tumor implantation. Animals were weighed dailyand tumors were measured 2 times weekly by caliper.

Toxicity and efficacy evaluation were performed as reported in example10.1.

Results

After a single administration at 5.0 and 2.5 mg/kg, DM4-SPDB-huMAb1_1was well tolerated and was active producing a ΔT/ΔC<0 and regressions ofthe initial tumor, volume of 100%, with 6/6 CR at 5 mg/kg and 4/6 CR at2.5 mg/kg. The dosages below 1.25 mg/kg was active with ΔT/ΔC=30%(p=0.0015) and no regressions. The lower dose 0.62 mg/kg yielded aΔT/ΔC=86% (Table 35, FIG. 24)

TABLE 35 Evaluation of the anti-tumor activity of DM4-SPDB-huMAb1_1against human primary invasive ductal carcinoma BRE-IGR-0159 in SCIDfemale mice Average body weight Route/ Dosage in Drug change in % MedianMedian % Tumor Dosage in mg/kg per death per mouse ΔT/ΔC of free mL/kgper injection Schedule (Day of at nadir in % regression Regressionssurvivors Biostatistic Agent injection (total dose) in days death) (dayof nadir) day 24 on day 27 Partial Complete day 49 p value 24 DM4-SPDB-IV 10 5.0 (5.0) 17 0/6 −1.7 (18) <0 100 6/6 6/6 6/6 p < 0.0001 huMAb1_1mL/kg 2.5 (2.5) 17 0/6 −3.0 (32) <0 100 6/6 4/6 3/6 p < 0.0001 1.25(1.25) 17 0/6 −3.3 (34) 30 — 0/6 0/6 0/6 p = 0.0015 0.62 (0.62) 17 0/6−8.1 (31) 86 — 0/6 0/6 0/6 p = 0.9232 Control 0/6  −13 (31) Tumordoubling time = 5.3 days. Tumor size at start of therapy was 80-270 mm³,with a median tumor burden per group of 144-153 mm³. Mice averageweight: Due to body weight heterogeneity (range: SAR428926 = 20.50-26.10g) dosages were adjusted to the individual body weights. Drugformulation: HGS buffer pH = 5.5. Statistical analysis: Dunnett's testversus control following a two-way Anova with repeated measuresperformed separately for each compounds on ranks of changes frombaseline. A probability less than 5% (p < 0.05) was considered assignificant.

Example 10.2.3: Evaluation of the Anti-Tumor Activity ofDM4-SPDB-huMAb1_1 Against Primary Human Lung Tumor LUN-NIC-0070 Materialand Method

DM4-SPDB-huMAb1_1 was evaluated at 4 doses against measurable primarycolon CR-LRB-010P tumors implanted s.c in female SCID mice. Controlgroups were left untreated. DM4-SPDB-chMAb2 was administered at 10, 5,2.5 and 1.25 mg/kg by an intravenous (IV) bolus injection, on day 35 at10, 5, 2.5 mg/kg and 49 at 1.25 mg/kg 19 post tumor implantation.Animals were weighed daily and tumors were measured 2 times weekly bycaliper.

Toxicity and efficacy evaluation were performed as reported in example10.1.

Results

DM4-SPDB-huMAb1_1 given at 10.0; 5.0 2.5 and 1.25 mg/kg was welltolerated. After a single administration at 10; 5 and 2.5 mg/kgDM4-SPDB-huMAb1_1 was active with a ΔT/ΔC<0 ((p<0.0001) and 100%complete regressions. The dosage below 1.25 mg/kg was active with 2.5mg/kg ΔT/ΔC<0 ((p<0.0001) and 4/6 PR (Table 36 a) and b), FIG. 25).

TABLE 36 Evaluation of the anti-tumor activity of DM4-SPDB-huMAb1 gainstprimary human lung tumor LUN-NIC-0070 in SCID female mice. a) Averagebody weight Route/ Dosage in Drug change in % Median Median % TumorDosage in mg/kg per death per mouse ΔT/ΔC of free mL/kg per injectionSchedule (Day of at nadir in % regression Regressions survivorsBiostatistic Agent injection (total dose) in days death)c (day of nadir)day 59 on day 59 Partial Complete at day 84 p value DM4-SPDB- IV/10 10(10) 35 0/6 −4.1 (64) <0 100 6/6 6/6 6/6 p < 0.0001 huMAb1 5 (5) 0/6^(a)) −4.9 (50) <0 100 5/5 5/5 5/5 p < 0.0001 2.5 (2.5) 0/6 −4.2(64) <0 100 6/6 6/6 5/6 p < 0.0001 controls 0/9 −2.0 (37) 0/6 0/6 0/6.Tumor doubling time = 8.8 days. Tumor size at start of therapy was96-218 mm³ with a median tumor burden per group of 132-138 mm³. Drugformulation: His-Gly-Sucrose buffer pH = 5.5; Statistical analysis:Dunnett's test versus control following a two-way Anova with repeatedmeasures performed separately for each compounds on ranks of changesfrom baseline. A probability less than 5% (p < 0.05) was considered assignificant b) Average body weight Route/ Dosage in change in % MedianMedian % Tumor Dosage in mg/kg per per mouse ΔT/ΔC of free mL/kg perinjection Schedule Drug at nadir in % regression Regressions survivorsBiostatistic Agent injection (total dose) in days death (day of nadir)day 59 on day 59 Partial Complete at day 84 p value DM4-SPDB- IV/10 1.25(1.25) 49 0/6 −4.3 (63) <0 (64 on 4/6 0/6 0/6 p < 0.0001 huMAb1 day 71)controls 0/6 −2.8 (56) 0/6 0/6 0/6 Tumor doubling time = 10.3 days.Tumor size at start of therapy was 118-220 mm³ with a median tumorburden per group of 171-176 mm³. Drug formulation: His-Gly-Sucrosebuffer pH = 5.5; Statistical analysis: Dunnett's test versus controlfollowing a two-way Anova with repeated measures performed separatelyfor each compounds on ranks of changes from baseline. A probability lessthan 5% (p < 0.05) was considered as significant.

Example 10.3: In Vivo Efficacy of DM4-SPDB-chMAb2 Example 10.3.1:Evaluation of the Anti-Tumor Activity of DM4-SPDB-chMAb2 Against PrimaryHuman Colon Adenocarcinoma CR-LRB-010P Material and Method

DM4-SPDB-chMAb2 was evaluated at 3 doses against measurable primarycolon CR-LRB-010P tumors implanted s.c in female SCID mice. Controlgroups were left untreated. DM4-SPDB-chMAb2 was administered at 10, 5and 2.5 mg/kg by an intravenous (IV) bolus injection, on day 19 posttumor implantation. Animals were weighed daily and tumors were measured2 times weekly by caliper.

Toxicity and efficacy evaluation were performed as reported in example10.1.

Results

DM4-SPDB-chMAb2 given at 10.0; 5.0 and 2.5 mg/kg was well tolerated.After a single administration at 10 and 5 mg/kg DM4-SPDB-chMAb2 wasactive with a ΔT/ΔC<0 ((p<0.0001) and 1/6 PR and ΔT/ΔC=10 (p<0.0001) andno regressions respectively. The dosage below 2.5 mg/kg produced aΔT/ΔC=68%. (Table 37, FIG. 26)

TABLE 37 Evaluation of the anti-tumor activity of DM4-SPDB-chMAb2against primary human colon adenocarcinoma CR-LRB-010P in SCID femalemice. Average body weight Route/ Dosage in Drug change in % Median %Dosage in mg/kg per death per mouse Median of mL/kg per injectionSchedule (Day of at nadir ΔT/ΔC regression Regressions BiostatisticAgent injection (total dose) in days death) (day of nadir) in % day onday Partial Complete p value (day) DM4-SPDB- IV 10 10 (10) 19 0/6 −1.6(20) <0 (31) 36 (31) 1/6 0/6 p < 0.0001 (31) chMAb2 mL/kg 5 (5) 19 0/6−2.4 (37) 10 (28) — 0/6 0/6 p < 0.0001 (28) 2.5 (2.5) 19 0/6 −2.5 (36)66 (28) — 0/6 0/6 p = 0.7711 (28) Control  0/10 −3.5 (37) — — 0/6 0/6 —Tumor doubling time = 6.0 days. Tumor size at start of therapy was88-277 mm³, with a median tumor burden per group of 138-157 mm³. Miceaverage weight range = 18.10-22.40 g dosages were adjusted to theindividual body weights. Drug formulation: His-Gly-Sucrose buffer pH =5.5; Statistical analysis: Dunnett's test versus control following atwo-way Anova with repeated measures performed separately for eachcompounds on ranks of changes from baseline. A probability less than 5%(p < 0.05) was considered as significant.

Example 10.3.2: Evaluation of the Anti-Tumor Activity of DM4-SPDB-chMAb2Against Human Primary Invasive Ductal Carcinoma BRE-IGR-0159 in SCIDFemale Mice Material and Method

DM4-SPDB-chMAb2 was evaluated at 3 doses against measurable primaryinvasive ductal carcinoma BRE-IGR-0159 tumors implanted s.c in femaleSCID mice. Control groups were left untreated. DM4-SPDB-chMAb2 wasadministered at 10, 5 and 2.5 mg/kg by an intravenous (IV) bolusinjection, on day 14 post tumor implantation. Animals were weighed dailyand tumors were measured 2 times weekly by caliper.

Toxicity and efficacy evaluation were performed as reported in example10.1.

Results

DM4-SPDB-chMAb2 given at 10.0; 5.0 and 2.5 mg/kg was well tolerated.After a single administration at 10, 5 and 2.5 mg/kg DM4-SPDB-chMAb2 wasactive with a ΔT/ΔC<0 ((p<0.0001) and 100% complete regressions at alldoses tested. (Table 38, FIG. 27)

TABLE 38 Evaluation of the anti-tumor activity of DM4-SPDB-chMAb2against human primary invasive ductal carcinoma BRE-IGR-0159 in SCIDfemale mice Average body weight Route/ Dosage in Drug change in % MedianMedian % Tumor Dosage in mg/kg per death per mouse ΔT/ΔC of BiostatisticFree Agent mL/kg per injection Schedule (Day of at nadir in % regressionRegressions p value survivor (batch) injection (total dose) in daysdeath) (day of nadir) day 24 on day 24 Partial Complete day 24 on day124 DM4-SPDB- IV 10 10.0 (10.0) 14 0/6 −5.2 (17) <0 100 5/5 5/5 p <0.0001 5/5 chMAb2 mL/kg 5.0 (5.0) 14 0/6 −6.9 (17) <0 100 6/6 6/6 p <0.0001 6/6 2.5 (2.5) 14 0/6 −4.2 (17) <0 100 6/6 6/6 p < 0.0001 4/6Control — — —  0/10 −15.9 (24)  — — 0/6 0/6 0/6 Tumor doubling time =2.9 days. Tumor size at start of therapy was 88-245 mm3, with a mediantumor burden per group of 120-135 mm3. Statistical analysis: Dunnett'stest versus control following a two-way Anova with repeated measuresperformed separately for each compounds on ranks of changes frombaseline. A probability less than 5% (p < 0.05) was considered assignificant.

Example 10.4: Efficacy of the Anti-Tumor Activity of DM4-SPDB-chMAb3Against Human Primary Invasive Ductal Carcinoma BRE-IGR-0159 in SCIDFemale Mice Material and Method

DM4-SPDB-chMAb3 was evaluated at 3 doses against measurable primaryinvasive ductal carcinoma BRE-IGR-0159 tumors implanted s.c in femaleSCID mice. Control groups were left untreated. DM4-SPDB-chMAb3 wasadministered at 5.0, 2.5 and 1.25 mg/kg by an intravenous (IV) bolusinjection, on day 16 post tumor implantation. Animals were weighed dailyand tumors were measured 2 times weekly by caliper.

Toxicity and efficacy evaluation were performed as reported in example10.1.

Results

DM4-SPDB-chMAb3 given at 5.0, 2.5 and 1.25 mg/kg was well tolerated.After a single administration at 5.0, 2.5 and 1.25 mg/kg DM4-SPDB-chMAb3was active with a ΔT/ΔC<0 ((p<0.0001) and 100% of regressions at alldose tested. (Table 39, FIG. 28)

TABLE 39 Evaluation of the anti-tumor activity of DM4-SPDB-chMAb3against human primary invasive ductal carcinoma BRE-IGR-0159 in SCIDfemale mice Average body weight Route/ Dosage in Drug change in % Median% Tumor Dosage in mg/kg per death per mouse Median of free mL/kg perinjection Schedule (Day of at nadir ΔT/ΔC regression Regressionssurvivors Biostatistic Agent injection (total dose) in days death) (dayof nadir) in % (day) on day 35 Partial Complete day 76 p value DM4-SPDB-IV 10 5.0 (5.0) 16 0/6 −2.8 (17) <0 (35) 100 6/6 6/6 6/6 p < 0.0001chMAb3 ml/kg 2.5 (2.5) 16 0/6 −3.2 (17) <0 (35) 100 6/6 5/6 5/6 p <0.0001 1.25 (1.25) 16 0/6 −1.7 (17) <0 (35) 100 6/6 6/6 2/6 p < 0.0001Control  0/10 −17.8 (32)  — 0/6 0/6 0/6 Tumor doubling time = 7.4 days.Tumor size at start of therapy was 88-221 mm³, with a median tumorburden per group of 135-151 mm³. Mice average weight range = 18.10-22.40g dosages were adjusted to the individual body weights. Drugformulation: His-Gly-Sucrose buffer pH = 5.5. Statistical analysis:Dunnett's test versus control following a two-way Anova with repeatedmeasures performed separately for each compounds on ranks of changesfrom baseline. A probability less than 5% (p < 0.05) was considered assignificant.

Example 11: In Vitro ADCC Activity

ADCC activity was evaluated using HCT116 huLAMP1 clone 8 (as describedin example 9) as target cells and human natural killer (NK) cells aseffector cells. A lactate dehydrogenase (LDH) release assay was used tomeasure cell lysis (R. L. Shields et al., 2001, J Biol Chem, 276:6591-6604).

Peripheral Blood Mononuclear Cells Isolation

Blood was diluted 2-3-fold with phosphate-Buffered Saline (PBS). Thirtyfive mL of diluted blood was carefully layered over 15 mL ofFicoll-Paque Plus (GE healthcare) in a 50 mL conical tube andcentrifuged at 400 g for 40 min at room temperature. The peripheralblood mononuclear cells (PBMC) were collected from the interface,transferred into a new conical 50 mL tube, and washed twice with PBS.

NK Isolation

According to Miltenyi NK cell isolation kit protocol (130-092-657,Miltenyi Biotech). The PBMC were suspended in NK-isolation buffer (40 μlof buffer for 10⁷ total cells), and then Biotin-Antibody Cocktail (10 μlfor 10⁷ total cells) was added to the cell suspension. TheBiotin-Antibody Cocktail contains biotinylated antibodies that bind tothe mononuclear cells, except for NK cells. The mixture was incubated at4° C. for 5 min, and then NK-isolation buffer (30 μl of buffer for 10⁷total cells) and NK cells MicroBead cocktail (20 μl for 10⁷ total cells)were added. The cell-antibody mixture was incubated for another 10 minat 4° C. Next, cells were washed (centrifugation at 400 g for 10 min)once with 50 mL of NK-isolation buffer, suspended in 1 mL ofNK-isolation buffer for 2.10E+8 cell and loaded on isolated by theautoMACS Pro Separator (Miltenyi) using the depletion program. Collectedand pooled negative fractions (containing NK cells) were washed once(centrifugation at 400 g for 10 min) and suspended at 2.5×10⁶/mL inRPMI-1640 supplemented with 10% fetal bovine serum, 2 mM of L-Glutamine,1% of penicillin/streptomycin, 1% Hepes, 1% Na-pyruvate and 1%non-essential amino-acids.

ADCC Protocol

10-fold serial dilutions, from 1.5×10⁻⁷ M to 1.5×10⁻¹⁷ M of testedantibody as well as isotypic control antibody were prepared in RPMI-1640medium supplemented with 0.1% BSA, 2 mM HEPES, pH 7 0.4 (denoted belowas RHBP medium). Triplicate of each antibody concentration weredistributed (50 μL/well) into a round bottom 96-well plate. HCT116huLAMP1 clone 8 cells were suspended at 0.075×10⁶ cells/mL in RHBPmedium and added to each well (100 μL/well) containing antibodydilutions. The plate containing target cells and antibody dilutions wasincubated for 10 min at room temperature. NK cells were washed andsuspended in RHBP medium at 0.75×10⁶ cells/mL, 50 μL of NK cells werethen added to each well, leading to a typically ratio of 5 NK cells to 1target cell. Control A consisted of wells containing only target cells(no antibody and no NK cells added) where RHBP medium (50 μL/well) wasadded instead of NK cells. Control B consisted of wells containing onlytarget cells (no antibody and no NK cells added) where 20 μL of TritonX-100 solution (RPMI-1640 medium, 10% TritonX-100) was added, todetermine the maximum possible LDH release of target cells. The mixtureswere incubated at 37° C. for 4 h, and then centrifuged for 10 min at1200 rpm, 100 μL of the supernatant was carefully transferred to a newflat-bottom 96-well plate. Freshly prepared LDH reaction mixture (100μL/well) from Cytotoxicity Detection Kit (Roche 11644793001) was addedto each well and incubated in dark at room temperature for 30 min.

The optical density of samples was measured at 490 mn (0D490). 100% oflysis corresponded to OD490 value of control B wells and 0% of lysis tothe OD490 value of the control A wells. The percent specific lysis ofeach sample was determined by the following formula: (0D490 sample−OD490of control A)/(0D490 control B−OD490 control A)*100

The samples containing only NK cells gave negligible OD490 readings.

Example 11.1: In Vitro ADCC Mediated by chMAb1, chMAb2 and chMAb3

The chMAb1, chMAb2 and chMAb3 antibodies specifically induced similarand potent ADCC activities as shown in FIG. 29. Isotype control antibodyhad no significant ADCC activity

Example 11.2: Variability of In Vitro ADCC

ADCC activities of chMAb1 or chMAb2 were evaluated for several batchesof purified NK, each batch correspond to an individual blood donor. Asdepicted in table 40, ADCC activities varied from one batch of NK cellsto the other. EC₅₀ values were estimated using BIOST@T-SPEED software.

TABLE 40 Maximum of ADCC and EC₅₀ for individual batches of isolated NKcells Highest ADCC value (% of maximal NK batch # LDH release) EC₅₀ (nM)Comment chMAb1 67125903091 36 0.76 67125626389 51 0.05 6712562616 44 NotEC₅₀ could not be determined as determined high plateau not reach forhighest concentration of MAb assayed 67130127373 33 0.009 67130127429 60Not EC₅₀ could not be determined as determined high plateau not reachfor highest concentration of MAb assayed 67130496259 20 0.6 6713049455233 0.2 chMAb2 67125903091 32 0.5 67125626389 52 0.1 6712562616 31 267130127429 61 Not EC₅₀ could not be determined as determined highplateau not reach for highest concentration of MAb assayed

Example 11.3: In Vitro ADCC Dependency on LAMP1 Antigen Density

Using the same NK batch, ADCC was analyzed for HCT116 huLAMP1 clonesdisplaying different antigen densities. As illustrated by data of FIG.30, antigen density >20 000 is required to lead to noticeable in vitroADCC activity.

Example 11.4: Comparison of In Vitro ADCC of chMAb1 and DM4-SPDB-chMAb1or chMAb2 and DM4-SPDB-chMAb2

DM4-SPDB conjugation did not significantly impacts ADCC activity ofchMAb1 or chMAb2 (FIGS. 31a and b ).

Example 11.5: In Vitro ADCC Mediated by huMAb1_1

ChMAb1 was included in the experiments a reference comparator. HuMAb1_1induced ADCC activity similar to chMAb1 as shown in FIG. 32.

Example 11.6: In Vitro ADCC Mediated by DM4-SPDB-huMAb1_1

HuMAb1_1 was included in the experiments a reference comparator.DM4-SPDB-huMAb1_1 induced ADCC activity similar to huMAb1 as shown inFIG. 33.

Example 12: In Vitro ADCP Activity

ADCP activity was evaluated using HCT116 huLAMP1 clones with differentLAMP1 antigen densities as target cells and human macrophages aseffector cells. HCT116 huLAMP1 clones were labeled by PKH67 fluorescentdye, macrophages were labeled by CD14-PC7 fluorescent dye.

Peripheral Blood Mononuclear Cells Isolation

The peripheral blood mononuclear cells (PBMC) were isolated as describedin example 11.

Monocytes Isolation

From isolated PBMC and according to Miltenyi monocytes cell isolationkit protocol (130-050-201, Miltenyi Biotech). Cells labeled byCD14-MicroBead were isolated using the positive selection program of theautoMACS Pro Separator (Milteny Biotech). Collected fraction (containingmonocytes cells) were suspended in 13.6 mL of RPMI-1640 supplementedwith 10% fetal bovine serum, washed once (centrifugation at 400 g for 10min) and suspended at a final concentration of 10⁶ cells/mL in 64 mL ofRPMI-1640 supplemented with 10% fetal bovine serum, 1% of heatinactivated human serum (AB; #14-490E), 2 mM of L-glutamine and 50 ng/mLof GM-CSF (Miltenyi Biotech; #130-093-866).

Macrophages Differentiation

The 64 mL of isolated monocytes were added to T75 flasks (NUNC;#156472), 10 mL per flasks. Flasks were put in a 37° C. 5% CO2 incubatorwhere cells were allowed to adhere for 8 days. RPMI-1640 supplementedwith 10% fetal bovine serum, 1% of heat inactivated human serum, 2 mM ofL-glutamine and 50 ng/mL of GM-CSF was changed after 4 days ofincubation.

ADCP Protocol

Macrophages were suspended by accutase (Invitrogen Stempro; #A111-0501), washed once (centrifugation at 400 g for 10 min) andsuspended in RPMI-1640 medium supplemented with 2% fetal bovine serumand 2 mM L-glutamine at a concentration of 1.5×10⁶ cells/mL for a ratioof 6/1 or 0.75×10⁶ cells/mL for a ratio of 3/1. 100 μL of suspendedmacrophages were distributed into a round bottom 96-well polypropyleneplate. 10⁷ of suspended target cells (HCT116 huLAMP1 clone 4; HCT116huLAMP1 clone 5; HCT116 huLAMP1 clone 8 or HCT116 huLAMP1 clone 12) werelabeled by PKH67 fluorescent dye following provider's procedure(SIGMA-ALDRICH; # MIDI67-1KT), then suspended at 5×10⁵ cells/ml inRPMI-1640 supplemented with 2% fetal bovine serum and 2 mM ofL-glutamine. 1/3 serial dilutions, from 9×10⁻⁸ M to 3×10⁻¹² M of testedhuMAb1_1 as well as isotypic control antibody were prepared in RPMI-1640medium supplemented with 2% fetal bovine serum. Duplicate of eachantibody concentration were distributed (150 μL/well) into a roundbottom 96-well polypropylene plate. 150 μL of PKH67-labeled target cellswere added to each well containing antibody dilutions. The platecontaining target cells and antibody dilutions was incubated for 15 minat 37° C. 100 μL of mixture (target cells+antibody) were added to the96-well plate containing macrophages which was then placed for 4 h to 17h in a 37° C. 5% CO2 incubator. Cells were suspended by accutase(Invitrogen stempro; # A111-0501), washed twice (centrifugation at 400 gfor 10 min) and suspended in buffer (PBS supplemented with 5% of heatinactivated human serum) containing CD14-PC7 antibody (Beckman Coulter;# PN A22331). After 20 minutes of incubation at 4° C., cells were washedonce and suspended in 250 μL of PBS, washed once and suspended in 50 μLof fixing paraformaldehyde solution (PFA 4% in PBS, USB; #19943).Samples were stored at 4° C. up to cytometry analysis.

Cytometry Analysis

Samples were analyzed using a MACSQUANT apparatus. Phagocytosis wasdetermined as double-labeled cells (PKH67 positive and PC7 positivecells). Typical data obtained are shown in FIG. 34.

Example 12.1: In Vitro ADCP Mediated by huMAb1_1

Two ratios of macrophages/HCT116 huLAMP1 clone 8 were analyzed. EC₅₀values were estimated using BIOST@T-SPEED software. HuMAb1_1 inducessignificant in vitro ADCP at a ratio macrophages/target cell of 3/1.Higher ratio, 6/1, did not lead to lower EC₅₀ or higher % ofphagocytosis as shown in Table 41.

TABLE 41 In vitro ADCP mediated by huMAb1_1 Ratio macrophages/ Batch ofMaximum of target cell macrophages EC₅₀ (nM) phagocytosis 3/167131617438 0.28 32 67131617470 0.56 39 67131354967 0.5 43 671307134950.47 33 67130713495 0.36 34 6/1 67130713495 0.98 41 67130713495 0.34 40

Example 12.2: In Vitro ADCP Dependency on LAMP1 Cell Surface Expression

ADCP was analyzed for HCT116 huLAMP1 clones displaying different antigendensities. The level of in vitro ADCP induced by huMAb1_1 is linked toantigen density of LAMP1 as maximum of phagocytosis decreased as LAMP1antigen density decreased as deducabkle from Table 42.

TABLE 42 In vitro ADCP dependency on LAMP1 cell surface expression MeanMaximum Antigen Mean EC₅₀ of phagocytosis Target cell density (nM) +/−STD (%) +/− STD HCT116 huLAMP1 300 000 0.33 +/− 0.21 45.5 +/− 15.5 clone5 HCT116 huLAMP1 100 000 0.43 +/− 0.11 36.2 +/− 4.7  clone 8 HCT116huLAMP1  20 000 0.73 +/− 0.17 34.0 +/− 9.1  clone 4 HCT116 huLAMP1  2500 0.65 +/− 0.49 8.7 +/− 4.1 clone 12

Example 12.3: In Vitro ADCP for huMAb1_negB

FCγRIIIa mediated phagocytosis was evaluated by assessing ADCP inducedby huMAb1_negB (described in example 7.2). As expected (macrophages notbeing strictly dependent on FCγRIIIa for activation), huMAb1_negB leadedto lower ADCP than huMAb1_1, however ADCP still occurred whenhuMAb1_negB was used. Typical data obtained are displayed in FIG. 35.

Example 12.4: In Vitro ADCP for huMAb1_negA

ADCP mediated by huMAb1_negA (described in example B7.2) was tested toevaluate antigen specificity mediated phagocytosis. As displayed in FIG.36, huMAb1_negA did not induce any ADCP, validating specificity of dataobtained for huMAb1_1.

Example 13: LAMP1 Gene Copy Number Change Materials and Methods:

Array-Based Oligonucleotide Comparative Genomic Hybridization (aCGH)

Genomic DNA was analyzed using the Human Genome CGH Microarray400k-(Agilent Technologies, Santa Clara, Calif., USA). Digestion,labeling and hybridization were performed using Agilent Oligo aCGH Bravoplatform protocols for Human CGH 2×400K microarrays. In all experiments,sex-matched DNA from a human well-characterized normal female (NA12878)or one well-characterized normal male (NA10858) was used as referenceDNA. The normal human genomic DNA used in these experiences iscommercialized by Coriell reference.

Oligonucleotide aCGH processing was performed as detailed in themanufacturer's protocol (version 6.2 Oct. 2009; http://www.agilent.com).The microarray required 600 ng of genomic DNA from the reference sampleand from the experimental sample. Array was scanned with an Agilent DNAMicroarray Scanner (G2565CA).

Data were extracted from scanned images and normalized using the FeatureExtraction software (v10.7.3.1, Agilent).

The log 2 ratio and segmentation were generated using Array Studiosoftware. Array Studio, Array Viewer and Array Server and all otherOmicsoft products or service names are registered trademarks ortrademarks of Omicsoft Corporation, Research Triangle Park, N.C., USA.

Centralization of the log 2 ratio distribution was verified andsegmentation was performed using the CBS algorithm (Olshen et al.;Biostatistics (2004), 5(4): 557). Aberration status calling wasautomatically performed for each profile according to its internal noise(variation of log 2 ratio values across consecutive probes on thegenome). All genomic coordinates were established on the UCSC humangenome build hg19 (Karolchik D et al. Nucleic Acids Res 2003, 31: 51).The value log Ratio and Copy Number Change, for each region or gene, wasintroduced in an internal database for subsequent analysis.

Gene Expression

The gene expression analysis was performed using a GeneChip Expression3′-Amplification Reagents Kit and U133Plus GeneChip arrays (Affymetrix,Santa Clara, Calif., USA), using the Expression Analysis Cia platforme.All data were imported into Resolver software (Rosetta Biosoftware,Kirkland, Wash., USA) for database management, quality controls andAnalysis. Each mRNA is represented by one or more qualifier. The valueof expression from each qualifier was downloaded in the Patient-DerivedTumor Xenograft Tumor Bank database (Tumor bank database) for analysis.

Animals

Animals were maintained in the animal facilities of each institutionfollowing standard animal regulation and strict health controls allowingtransfer between members of the consortium. Swiss-nude and CB17-SCIDfemale mice, as well as NIH-nude rats were bred at Charles River France(Les Oncins, France). Mouse weights were over 18 g and rat weights wereover 160 g at the start of experiments. Their care and housing were inaccordance with institutional guidelines, as well as national andEuropean laws and regulations as put forth by the French Forest andAgriculture Ministry and the standards required by the UKCCCRguidelines.

Sample Preparation

Frozen fragments were cut in a cryostat at −20° C. then beginning andend sections were stained with HES (Haematoxylin-Eosin-Saffron) forhistological control and evaluation of tumor cell percentage bypathologists. Genomic DNA was extracted according to QIAamp DNA Kitprotocols v01 (Qiagen, Hilden, Germany). Total RNA was extracted fromtumor samples and purified with an RNAeasy kit (Qiagen), using the RNAextraction Qiagen protocols v01.

Molecular Characterization of PDX

The molecular characterization (CGH, RNA expression and IHC) of eachtumoral model of PDX was performed using the same Xenografts passage.

Immunohistochemistry

To associate genomic copy number aberration of LAMP1 gene and its region(13q34) with changes in the protein levels (Strong, medium, Faint andNegative) of the membrane localization of LAMP1 on PDXs (Frozen-Oct),specific staining (mAb1) was carried out on the same passage of PDX usedfor CGH and RNA expression characterization.

After avidin and biotin blocking (Endogenous Block, Ventana, 760-050),frozen sections (Frozen-OCT format) were incubated with murinemonoclonal antibody MAb1 (final concentration 1 μg/mL (for humansamples) and 1 and 5 μg/mL (for monkey samples) in Phosphate BufferSaline, PBS) for 32 min at 37° C. A postfixation step withglutaraldehyde (0.05% in NaCl 0.9% w/v) for 4 min was done. Thesecondary goat anti-mouse IgG2a-biotinylated was incubated for 12 min at37° C. (Southern Biotech, Ref 1080-08, dilution 1/200 in Ventana'sdiluent). Immunostaining was done with DAB Map chromogenic detection kitaccording to manufacturer's recommendations. A counterstaining step wasapplied to the cryostat sections with hematoxylin II (790-2208, VentanaMedical Systems, Inc USA) and bluing reagent was applied for 4 min(760-2037). Stained slides were dehydrated and coverslipped withCoverquick 2000 mounting medium (Labonord, Ref 05547530).

The negative controls used in this study consisted in omission ofprimary antibody and the use of IgG2a isotype (final concentration 1μg/mL in PBS).

For data analysis sections immunostained with purified murine antibodyMAb1 were scanned and digitized at a magnification of ×20 using ScanScope XT system (Aperio Technologies, Vista Calif.). Digitized imageswere then captured using Image Scope software (v10.2.2.2319 Aperio,Technologies).

The positive samples were scored with a scale of intensity from 1 to 3.Ranges of intensities were described as negative (0), weak (1), moderate(2) and strong (3). Cell frequency was the percentage of immunostainedcells and was estimated by the histologist observation as a median bysample. The cell frequency was ordered in 5 categories: 1 (0-5%), 2(6-25%), 3 (26-50%), 4 (51-75%) and 5 (76-100%).

A global expression was calculated according the Allred Score (AS)description. AS was obtained by adding the intensity and the proportionscores to obtain a total score that ranged from 0-8. The AS was reportedas a percent of the maximum global score and ranged in 5 categories:very low (0-25%), weak (26-50%), moderate (51-75%) and high (75-100%).The prevalence was defined as the percent of positive cases for theindication.

Basic descriptive statistics were calculated with Microsoft Excel 2003.For each indication, number of cases, positive cases number, prevalence,intensity score mean, frequency mean and Allred score were described.

Statistical Analyses

In order to study the relation between the mRNA expression and the LAMP1gene change (gain or amplification), we applied a Student test on PDXdata to compare the mRNA expression levels of the tumor PDX with orwithout Copy Number change. We also determined the correlation betweenmRNA expression levels of the CRC PDXs and their respective genomic copynumber variation of LAMP1 gene and their region (13q34) by a Personcorrelation test.

In addition, a correlation analysis using a larger set of colorectalpatients tissues samples (n=574) from the TCGA (The Cancer Genome Atlas)database, was performed between mRNA expression normalized and CopyNumber using a Spearman correlation test. In order to study theassociation of LAMP1 expression or no expression at the plasma membraneof PDX tumors cells determined by IHC analysis and the Copy Numberfactor change or no change, a Cochran-Mantel-Haenszel statistics wasperformed. For colon PDX the test was performed using IHC score(Negative-Faint, Medium and Strong) versus Copy Number (CN<2.5 andCN≥2.5). For lung and stomach PDXs, a stratified Cochran-Mantel-Haenszelstatistics was performed using IHC score (Negative-Faint, Medium-Strong)versus Copy Number (CN<2.5 and C≥2.5). The statistical analyses wereconducted using SAS 9.2; SAS Institute Inc. and Everstat V6 (Sanofibased on SAS 9 SAS Institute Inc.).

Bioinformatics Analyses: Copy Number Changes in the TCGA (the CancerGenome Atlas)

DNA samples are analyzed using the GISTIC (Genomic Identification ofSignificant Targets in Cancer) methodology (Beroukhim, R. et al.; Nature2010 463, 899; Beroukhim, R. et al.; Proc Natl Acad Sci USA (2007); 104,20007). Briefly, each marker is scored according to the mean amplitudeand frequency of focal amplification across the dataset, andsignificance values are computed by comparing to the distribution ofscores obtained by random permutation of the markers across the genome.Significant peak regions of amplification (or deletion) are identifiedusing an iterative peel-off procedure that distributes the scoreassociated with amplified (or deleted) segments among all peaks thatoverlap them (weighted according to each peak's score) until no newregion crosses the significance threshold of q-value ≤0.25 on eachchromosome. Finally, by taking into account the auto-correlation withinthe GISTIC score profiles, a confidence interval is computed for eachpeak region that is predicted to contain the true driver gene or geneswith at least 99% probability (TCGA Network. Nature 2008; 455: 1061).

The gene-based calls from GISTIC output were used. Genes were defined aspossessing deep deletions, shallow deletions, neutral copy number, lowgain, and high gain using specific thresholds, as follows. High gainsare log 2 ratios that exceed 1.32; low gains are from 0.3 to the highgain threshold; neutral segments have copy numbers between −0.5 to 0.3;shallow losses have copy numbers between −0.5 and the deep deletionthreshold; and deep deletions have copy numbers that are below −0.737.

Example 14: LAMP1 Gene Copy Number Change Using CRC PDXs Tumors Samples

A total of 61 Colon tumor PDX were analyzed using whole genomehigh-density aCGH 400K-oligonucleotide arrays. As indicated in Table 44,6 out of 61 (9.8%) colorectal cancer PDXs displays a high-level LAMP1gene amplification (i.e: CN ≥5 or log 2 ratio ≥1.32) and 57.4% shows aLAMP1 gene gain (i.e: 2.5≤CN <5 or 0.32≤ log 2 ratio <1.32).

Using Lung PDXs Tumor Samples

A total of 35 Lung tumor PDXs were analyzed using whole genomehigh-density aCGH 400K-oligonucleotide arrays. As depicted in Table 45;one Lung tumor PDX (3%) studied displays a high-level amplification ofLAMP1 gene (CN=9.26) and 26% shows a LAMP1 gene gain (i.e: 2.5 CN ≤5 or0.32≤ log 2 ratio <1.32).

Using Commercial Tumor DNA Tissues Samples

The CGH analysis was performed also using whole genome high-density aCGH400K-oligonucleotide arrays in the esophageal tumor DNA samples(Asterand). As indicated in Table 46 there is a gain or amplification ofthe LAMP1 gene in 2 out of 46 (4.3%) esophageal tumor samples studied,one of these (2%) shows a focal amplification of LAMP1 equal to 39.81copy. This high level and focal amplification of LAMP1 is detected in anAsian female (ES01_F12), 64 years old; the biopsy contains 80% of tumorcells.

Using Patient Tumor Samples by the Cancer Genome Atlas (TCGA) Data

Following analyses of Copy number changes were performed using the TOGA(The Cancer Genome Atlas) data. Using a larger set of colorectal samples(n=574); the results are extremely similar with those obtained usinginternal data (CRC PDX). Colorectal and rectum human adenocarninomaanalysis (the Cancer Genome Atlas) discloses 14.4% high amplification(HighAmp). Subsequent Copy Number Alteration analyses using TOGA datawere performed to search other tumor types for which LAMP1 wasamplified. In summary, LAMP1 DNA gene low-level gains (LowAmp; Log 2Ratio=0.3<LowAmp <1.32) and high-gain (amplifications) (HighAmp, Log 2Ratio ≥1.32 (theoretically, overall 5.0 copies or more)) is detected in28 tumor types, including Colorectal adenocarcionoma, Stomach, Liver,Lung (Adenocarcinoma and Squamous), Breast (Basal, BRCA, LUMA, LUMB andHER2), Ovary, Head & neck, Kidney (Kidney Chromophobe, Kidney RenalClear Cell Carcinoma, Kidney Renal Papilliary Cell Carcinoma, Cervicalsquamous Cell, Pancreatic, Prostate, Bladder urothelial, Glioma (Lowgrade glyoma and Glioblastoma multiform), Uterus, Thyroid, Leukemia,Lymphoma, Esophageal, Melanoma and Soft tissue sarcoma, LAMP1 high gainor amplification data of 12 of these tumor types including colorectaladenocarcionoma, stomach, liver, lung, breast, ovary, head and neck,cervical squamous cell, glioblastoma, glioma, uterus, thyroid and softtissue sarcoma are displayed in Table 43 and FIG. 10A.

TABLE 43 LAMP1 Genomic Alteration Summary: 18 TCGA tumor types AverageAverage Number % of <Log2> % of <Log2> of HighAmp HighAmp LowAmp LowAmpCases Tumor Type Tumor 0.000 0.000 16.071 0.688 56 BLCA BladderUrothelial Carcinoma 1.578 2.084 9.587 0.626 824 BRCA Breast InvasiveCarcinoma 1.471 1.347 10.294 0.504 68 CESC Cervical Squamous Cell 14.4601.892 41.463 0.714 574 COADREAD Colon and Rectum Adenocarcinoma 0.5364.495 1.964 0.642 560 GBM Glioblastoma 0.694 4.058 11.111 0.526 288 HNSCHead and Neck Squamous Cell 0.000 0.000 4.090 0.516 489 KIRC KidneyRenal Clear Cell 0.000 0.000 13.333 0.673 75 KIRP Kidney RenalPapilliary Cell 0.694 2.018 2.083 0.462 144 LGG Lower Grade Glioma 1.7543.268 15.789 0.620 57 LIHC Liver Hepatocellular carcinoma 1.132 1.6617.925 0.471 265 LUAD Lung adenocarcinoma 1.418 4.565 6.383 0.581 282LUSC Lung squamous cell 1.792 2.061 15.950 0.601 558 OV Serous Ovarian0.000 0.000 7.143 0.329 14 PAAD Pancreatic adenocarinoma 0.000 0.0002.000 0.475 100 PRAD Prostate Adenocarcinoma 2.273 1.430 23.485 0.517132 STAD Stomach Adenocarcinoma 0.000 0.000 0.877 0.450 228 THCA Thyroidadenocarcinoma 0.234 2.780 7.009 0.659 428 UCEC Uterine CorpusEndometrioid Carcinoma Log2 = 1.32 ≤ HighAmp < ∞; Log2 = 0.32 < LowAmp <1.32Genomic Definition of LAMP1 (13q34) Change (Gain/Amplification) on thePDXs

LAMP1 gene gain or amplification can be related to a focal somatic gainor amplification, a somatic large region gain or amplification on 13 q,a somatic chromosome duplication, a somatic chromosome triplication orpolyploidy.

In colon tumor PDX, the LAMP1 DNA gain or amplification is included in alarger amplicon involving: CUL4A, LAMP1, TFDP1, and GAS6. As show in theTable 44, for the Colon cancer PDX, the mean size segments are 8489.5 kband 49292.7 kb for Amplification and gain, respectively. The minimumregion involves 454 kb, which starts at base 113319683, ends at base115107245 and contains others genes than LAMP1: ADPRHL1, CUL4A, DCUNID2,GRTP1, LOC100130463, PCID2, PRO7, TFDP1, TMCO3 and F10. Most of DNA gainor amplification contains at least the genes: ADPRHL1, ΔTP11A, ΔTP4B,CUL4A, DCUN1D2, F10, F7, FAM70B, FLJ41484, FLJ44054, GAS6, GRK1, GRTP1,LAMP1, LINC00552, LOC100128430, LOC100130463, LOC100506063,LOC100506394, MCF2L, MCF2L-AS1, PCID2, PROZ, RASA3, TFDP1 andTMCO3C13orf35. The largest gain region covers 95.8 Mb(19,296,544-115,107,245).

TABLE 44 Description analysis of LAMP1 Copy number analysis of studiedgroups (LAMP1 Amplification, Gain, Diploid and Heteroloss (Complete orpartial loss of one allele of LAMP1 gene) on Colon cancer PDX.Descriptive statistics for parameter Segment (size in kb) StandardStatus N Mean Deviation Hetloss 1 41152 . Diploid 19 65768.5 31068.78Gain 35 49292.7 37513.6  Amplification 6 8489.5 13016.03

In Lung Tumors PDX (Table 45), the mean of size segments is 14966.4 kbfor gain. The minimum region covers 1186 kb.

TABLE 45 Description analysis of LAMP1 Copy number analysis of studiedgroups (LAMP1 Amplification, Gain, Diploid, Deletion and Heteroloss(Complete or partial loss of one allele of LAMP1 gene) on Lung cancerPDX. Descriptive statistics for parameter Segment (size in kb) StandardStatus N Mean Deviation Deletion 1 1460 . Hetloss 7 44592.4 39478.32Diploid 17 26744.9 37016.96 Gain 9 14966.4 31033.29 Amplification 1 4874.Genomic Definition of LAMP1 (13q34) Gain on Esophageal Human TumorCancer

In the Esophageal cancer DNA samples (Asterand), the LAMP1 gain oramplification is also including in a large amplicon, the largest gainregion involves 4523 kb (110584050-115107245) and the smallest regionpresent a LAMP1 amplification (Chr13q34) equal to 39.81 copy number.This focal amplification of LAMP1 covers 378 kb and includes 10 genes:ADPRHL1, CUL4A, F10, F7, GRTP1, LAMP1, LOC100130463, PCID2, PROZ andMCF2L.

TABLE 46 Description analysis of LAMP1 Copy number analysis of studiedgroups (LAMP1 Amplification, Gain, Diploid and Heteroloss (Complete orpartial loss of one allele of LAMP1 gene) on Eosophagus cancer tissues.Descriptive statistics for parameter Segment (size in kb) StandardStatus N Mean Deviation Loss 5 70415 29317.91 NoChange 39 48109.437656.46 Gain 1 4523 . Amplification 1 378 .

Example 15: Relation Between LAMP1 Gene Copy Number and mRNA GeneExpression

Analyses of the mRNA Expression Level by Gene Expression Profile and theCopy Number Change at LAMP1 Region

In addition to the analysis of LAMP1 Copy Number Change (amplificationand gain), using the CRC tumors PDX, the correlation between LAMP1amplification was evaluated by CGH analysis and LAMP1 expression byusing mRNA (Affymetrix technology). Results from the mRNA analysis,using Pearson correlation test (Table 47) indicated high correlation((r)=0.59; p<0.0001) between LAMP1 Copy Numbers and LAMP1 mRNAexpression levels (FIG. 8A). For the Colon tumors PDX, a Student test isperformed to compare LAMP1 gene copy number (with or withoutgain/amplification) and LAMP1 mRNA expression. mRNA expression analysiswas performed using Affymetrix technology (Table 48 and FIG. 8B).

TABLE 47 LAMP1: Copy Number Alteration Data and correlation with mRNAdata on CRC PDX Tumor Total of Number of Number of Number of Number ofCorr with P Type models Gain cases gain cases >4.5 diploid Hetloss mRNA(r) values CRC 58 45161.2 +/− 30710.7 +/− 66942.5 +/− 91670 +/− 0.59<0.0001 PDX 6987.02(Kb) 11123.32 (Kb) 6795.47 (Kb) (n = 1) (n = 29) (n =9) (n = 19) (<1%) (50%) (~15.5%) (~33%)

TABLE 48 Student t-test of mRNA expression for factor copy numberStudent t-test for factor CopyNumber Mean +/− SEM t Parameter <2.5 ≥2.5DF value p RNA 11785.30 +/− 14987.09 +/− 55 −4.04 p = Intensity 496.501(n = 18) 486.708 (n = 39) 0.0002 If p < 0.05, the factor has significantinfluence on parameter

The mRNA expression is significantly higher for LAMP1 Copy Numberschange (CN2.5).

The correlation analysis, using Pearson test between LAMP1 amplificationby CGH analysis and LAMP1 expression by using mRNA, shows asignificantly correlation between these two parameter studied.

As shown in FIG. 8a , the group with LAMP1 high amplification (Amp)shows higher mRNA expression levels than groups with LAMP1 lowamplification (Gain), Diploid and Hetloss.

The correlation analysis using a larger set of colorectal patientstissues samples (n=574) from the TCGA (The Cancer Genome Atlas) data,disclosed 14.4% amplification that correlates with mRNA expression((r)=0.57; p<0.0001), this result is extremely similar with thatobserved on the CRC PDX.

Moreover, using the same dataset, a significant correlation of LAMP1copy number change and mRNA expression level was evidenced for: BladderUrothelial Carcinoma (BLCA), Breast Invasive Carcinoma (BRCA), Lungadenocarcinoma (LUAD), Lung squamous cell (LUSC) and Ovary (OV) (FIG.9).

Example 16: LAMP1 Copy Number Variation and its Impact on the LAMP1Protein Cell Membrane Expression Level Association of LAMP1 Copy NumberChange and the Protein Cell Membrane Expression Level Detected byImmunohistochemistry (IHC).

In addition to the analysis of LAMP1 gain and its relation with theLAMP1 RNA expression, we also evaluated association of LAMP1 copy numberchange (gain or amplification) to cell membrane LAMP1 proteinlocalization, using IHC expression scoring (strong, medium, faint andnegative) with antibody mAb1 for colon, lung and stomach tumor PDXs.

As shown in tables 49 and 50 below, and FIG. 11, analysis of IHC cellmembrane expression in colon, lung and stomach PDXs samples shows thatLAMP1 protein is expressed in the membrane cells in 39 out of 95 PDXs(41.1%) models studied; 33 of these PDXs samples positive for LAMP1membrane expression (33 out of 39; 85%) present also LAMP1 gain oramplification, most of these are Colon PDX.

TABLE 49 Frequency of LAMP1 Copy Number data and IHC scoring data ofcolon tumor PDX Copy Number IHC Frequency Neg_Faint Medium Strong Total<2.5 15 4 0 19 ≥2.5 13 18 10 41 Total 28 22 10 60

TABLE 50 Frequency of LAMP1 Copy number data and IHC scoring data oflung and stomach tumor PDXs Copy Number IHC Frequency Tumor TypeNeg_Faint Medium_Strong Total <2.5 Lung 9 2 11 Stomach 11 0 11 ≥2.5 Lung5 1 6 Stomach 3 4 7

The association between IHC membrane expression and the copy numberchange was studied using Cochran-Mantel-Haenszel statistics (Tables 51and 52).

TABLE 51 Cochran-Mantel-Haenszel statistics of LAMP1 IHC membraneexpression by the Copy Number of LAMP1 in the Colon PDX tumor samples.Cochran-Mantel-Haenszel Statistics (Based on Rank Scores) AlternativeHypothesis DF Value Prob Nonzero Correlation 1 12.4418 0.0004

In Colon tumor PDX, the association between LAMP1 IHC membraneexpression and Copy Number of LAMP1 is significant.

TABLE 52 Cochran-Mantel-Haenszel statistics of LAMP1 IHC membraneexpression by the Copy Number of LAMP1 in Lung and Stomach PDX tumorsamples. Cochran-Mantel-Haenszel Statistics (Based on Rank Scores)Alternative Hypothesis DF Value Prob Nonzero Correlation 1 4.5416 0.0331

After adjusting for tumor type, the association between LAMP1 IHCmembrane expression and Copy Number of LAMP1 is significant.

We conclude that the level of LAMP1 cell surface localization (Strongand medium) is associated with copy number change on tumor PDX samples.Most of cell surface localization of LAMP1 appears to be a consequenceof LAMP1 gain or amplification.

TABLE 53 Table summarizing LAMP1 gene gain and LAMP1 expression SegmentLog2 ratio Membrane- Sample Name Indication Status Class (size in kb)(Mean) Copynumber IHC_Score IHC_Level2 Expression LUN-NIC-0070 LungAmplification Gain 4874 2.21 9.26 ** Medium Yes CR-LRB-0010-P ColonAmplification Gain 1029 2.15 8.88 *** Strong Yes CR-LRB-0011-M ColonAmplification Gain 34252 1.78 6.85 *** Strong Yes IMM-COLO-0010 ColonAmplification Gain 454 1.75 6.73 ** Medium Yes CR-IGR-0002-C ColonAmplification Gain 4451 1.62 6.14 ** Medium Yes CR-IC-0029-P ColonAmplification Gain 9080 1.58 5.99 *** Strong Yes IMM-COLO-0020 ColonAmplification Gain 1671 1.41 5.32 neg neg No CR-IC-0028-M Colon GainGain 95810 1.27 4.84 ** Medium Yes CR-LRB-0017-P Colon Gain Gain 564971.27 4.83 neg neg No CR-IGR-0025-P Colon Gain Gain 14032 1.27 4.82 **Medium Yes CR-IGR-0002-P Colon Gain Gain 59574 1.23 4.69 ** Medium YesGAS0232 Stomach Gain Gain 1787 1.19 4.57096889 *** Strong YesCR-IGR-0052-M Colon Gain Gain 17965 1.16 4.47 ** Medium YesIMM-COLO-0006 Colon Gain Gain 95810 1.04 4.12 ** Medium Yes CR-IC-0010-PColon Gain Gain 92308 1.04 4.11 neg neg No CR-IGR-0007-P Colon Gain Gain85070 0.99 3.97 *** Strong Yes CR-IGR-0047-P Colon Gain Gain 45330 0.973.91 ** Medium Yes CR-LRB-0013-P Colon Gain Gain 1575 0.89 3.71 **Medium Yes LUN-NIC-0004 Lung Gain Gain 4305 0.89 3.7 neg neg NoCR-IGR-0014-P Colon Gain Gain 95810 0.89 3.7 * Faint No CR-IGR-0016-PColon Gain Gain 20181 0.82 3.54 ** Medium Yes CR-LRB-0019-C Colon GainGain 941 0.8 3.49 *** Strong Yes LUN-NIC-0040 Lung Gain Gain 5329 0.793.46 neg neg No LUN-NIC-0047 Lung Gain Gain 1560 0.77 3.42 neg neg NoCR-IC-0007-M Colon Gain Gain 25657 0.76 3.39 * Faint No CR-IC-0006-MColon Gain Gain 4915 0.74 3.33 neg neg No CR-IC-0008-P Colon Gain Gain19106 0.71 3.27 ** Medium Yes CR-IGR-0048-M Colon Gain Gain 95810 0.713.26 * Faint No CR-LRB-0014-P Colon Gain Gain 16868 0.69 3.22 ** MediumYes SA-STO-0073 Stomach Gain Gain 3160 0.67 3.19 *** Strong YesCR-IGR-0008-P Colon Gain Gain 523 0.64 3.12 neg neg No GAS0081 StomachGain Gain 209 0.63 3.100283186 neg neg No SA-STO-0043 Stomach Gain Gain1984 0.61 3.05 ** Medium Yes CR-IGR-0009-P Colon Gain Gain 73073 0.59 3*** Strong Yes CR-IGR-0038-C Colon Gain Gain 14246 0.58 2.99 ** MediumYes CR-LRB-0009-C Colon Gain Gain 54677 0.55 2.93 ** Medium Yes GAS0080Stomach Gain Gain 1773 0.546 2.920671295 ** Medium Yes CR-IGR-0023-MColon Gain Gain 95810 0.54 2.91 * Faint No IMM-COLO-0004 Colon Gain Gain95810 0.47 2.78 *** Strong Yes GAS0832 Stomach Gain Gain 57217 0.472.767915691 neg neg No CR-IC-0005-P Colon Gain Gain 95810 0.47 2.76 ***Strong Yes LUN-NIC-0002 Lung Gain Gain 1590 0.45 2.73 neg neg NoIMM-COLO-0023 Colon Gain Gain 10757 0.45 2.72 *** Strong YesCR-IC-0020-P Colon Gain Gain 4364 0.44 2.71 ** Medium Yes CR-IC-0019-PColon Gain Gain 95810 0.44 2.71 * Faint No CR-IC-0013-M Colon Gain Gain39036 0.43 2.69 ** Medium Yes GAS0819 Stomach Gain Gain 1994 0.412.665858119 neg neg No CR-IGR-0011-C Colon Gain Gain 45835 0.38 2.6 **Medium Yes CR-IC-0009-M Colon Gain Gain 45566 0.38 2.6 *** Strong YesCR-LRB-0022-P Colon Gain Gain 4874 0.37 2.59 * Faint No CR-IGR-0034-PColon Gain Gain 16139 0.36 2.56 ** Medium Yes LUN-NIC-0051 Lung GainGain 95810 0.33 2.51 neg neg No CR-LRB-0007-P Colon Gain Gain 94994 0.322.5 * Faint No CR-IC-0016-M Colon Gain Gain 94661 0.32 2.5 * Faint NoCR-IC-0032-P Colon NoChange NoGain 41073 0.3 2.47 * Faint NoLUN-NIC-0011 Lung NoChange NoGain 1560 0.3 2.46 neg neg No CR-IC-0025-MColon NoChange NoGain 57313 0.3 2.46 ** Medium Yes CR-IC-0002-P ColonNoChange NoGain 86532 0.29 2.44 ** Medium Yes LUN-NIC-0006 Lung NoChangeNoGain 1587 0.28 2.42 ** Medium Yes CR-IC-0021-M Colon NoChange NoGain57313 0.23 2.35 * Faint No LUN-NIC-0034 Lung NoChange NoGain 5334 0.222.33 neg neg No LUN-NIC-0041 Lung NoChange NoGain 1753 0.2 2.29 neg negNo GAS0773 Stomach NoChange NoGain 46737 0.17 2.25 neg neg NoCR-IC-0003-P Colon NoChange NoGain 95810 0.16 2.24 * Faint NoCR-IC-0004-M Colon NoChange NoGain 14209 0.14 2.2 * Faint NoCR-IGR-0032-P Colon NoChange NoGain 95810 0.1 2.15 * Faint NoSA-STO-0014 Stomach NoChange NoGain 7816 0.1 2.14 neg neg No GAS0720Stomach NoChange NoGain 4061 0.099 2.14 neg neg No CR-IGR-0029-P ColonNoChange NoGain 57313 0.07 2.11 neg neg No IMM-COLO-0018 Colon NoChangeNoGain 4936 0.06 2.09 neg neg No IMM-COLO-0008 Colon NoChange NoGain95810 0.04 2.05 * Faint No IMM-COLO-0001 Colon NoChange NoGain 958100.03 2.05 neg neg No LUN-NIC-0060 Lung NoChange NoGain 33461 0.02 2.03neg neg No GAS0517 Stomach NoChange NoGain 1739 0.001 2.001 neg neg NoSTO-IND-0006 Stomach NoChange NoGain 95810 0 2 neg neg No SA-STO-0039Stomach NoChange NoGain 14361 0 2 neg neg No CR-LRB-0018-P ColonNoChange NoGain 62941 −0.02 1.97 neg neg No CR-LRB-0003-P Colon NoChangeNoGain 57313 −0.02 1.97 ** Medium Yes SA-STO-0024 Stomach NoChangeNoGain 7831 −0.03 1.96 neg neg No CR-LRB-0004-P Colon NoChange NoGain57313 −0.03 1.96 ** Medium Yes CR-IGR-0012-P Colon NoChange NoGain 78930−0.03 1.96 * Faint No IMM-COLO-0009 Colon NoChange NoGain 95810 −0.041.95 * Faint No CR-IC-0022-P Colon NoChange NoGain 7897 −0.08 1.89 *Faint No GAS0928 Stomach NoChange NoGain 95810 −0.107 1.86 neg neg NoLUN-NIC-0001 Lung NoChange NoGain 1567 −0.19 1.75 neg neg No GAS0680Stomach NoChange NoGain 1217 −0.31 1.61 neg neg No LUN-NIC-0007 LungNoChange NoGain 1481 −0.32 1.6 neg neg No CR-IGR-0003-P Colon NoChangeNoGain 91670 −0.34 1.58 * Faint No LUN-NIC-0066 Lung Loss NoGain 20716−0.53 1.38 neg neg No GAS0248 Stomach Loss NoGain 57313 −0.58521.333113855 neg neg No LUN-NIC-0081 Lung Loss NoGain 52860 −0.59 1.33neg neg No CR-IGR-0043-P Colon Loss NoGain 41152 −0.83 1.12 neg neg NoLUN-NIC-0030 Lung Loss NoGain 95810 −0.85 1.11 ** Medium Yes GAS0707Stomach Loss NoGain 3462 −0.9479 1.036772962 neg neg No LUN-NIC-0033Lung Deletion NoGain 1460 −1.16 0.89 neg neg No

Example 17:—Specific Peptide and mAb to Detect LAMP1 MembraneReinforcement on FFPE Tumor Tissue by Immunohistochemistry (IHC)

IHC analysis of tumor tissues from biobanks or from hospitals before orduring patient treatment is routinely done with formalin-fixedparaffin-embedded (FFPE) samples. Although commercially available mAbsand the three mAbs described previously (MAb1, MAb2 and MAb3) can allowintracellular detection of LAMP1 and some of them, including MAb1, 2 and3, LAMP1 membrane reinforcement in frozen-OCT and AFA (Alcohol FormalinAcetic acid Fixative) sample format, none can lead to the detection ofLAMP1 reinforcement in FFPE format. One of the reasons is probably theeffect of the formalin fixative combined to the complexity of theprotein. Samples processed in frozen OCT or AFA are not routinelyprepared in hospitals. Therefore, there is a need to have a mAb thatwould allow complete and fast coverage of the FFPE tumor biobanks andhospital samples.

It is shown in the examples below that it was possible to overcome thedifficulties by identifying a peptide (peptide 4) located in the secondluminal domain at positions 360 to 375 of SEQ ID NO: 24, and having theamino acid sequence of SEQ ID NO: 82. Said peptide permitted theobtention of rabbit polyclonal antibodies and mouse monoclonal antibodythat led to the detection of LAMP1 membrane reinforcement in FFPEtissues.

TABLE 54 List of antiLAMP1 mAb tested and showing no LAMP1 membranereinforcement on FFPE tissues by IHC Species clone number MAbs obtainedfrom the following supplier Epitomics Rabbit ERP4204 Novus BiologicalsMouse B-T47 Biolegend Mouse H4A3 United States Biol Mouse 5K76 SantaCruz Mouse E-5 Santa Cruz Mouse H5G11 Biorbyt Mouse monoclonal BiorbytRabbit monoclonal MAbs described in this application MAb1 Mousemonoclonal MAb2 Mouse monoclonal MAb3 Mouse monoclonal

Example 17.1: Production of Rabbit Polyclonal Antibodies that LED toLAMP1 Membrane Reinforcement on FFPE Tumor Tissues

This example describes the selection of peptides in the human LAMP1luminal domains, the generation of polyclonal antibodies and the IHCscreening. It demonstrates the feasibility to obtain polyclonalantibodies that allow the detection of LAMP1 membrane reinforcement onformalin-fixed paraffin-embedded tissues when using the specific peptide(“peptide 4”) of SEQ ID NO:82 corresponding to the amino acids atpositions 360 to 375 on the human LAMP1 sequence of SEQ ID NO:24.

Example 17.1.1: Rabbit Immunisation with Peptides or Soluble LAMP1Protein. Purification of Polyclonal Antibodies Peptide Preparation:

Peptides of 15-16 amino acids were selected within the two luminaldomains without a N-glycosylation site and no internal cysteine. A totalof four peptides were chemically synthesised and coupled to the KeyholeLimpet Hemocyanin (KLH) carrier protein. When needed, a terminalcysteine was previously added to the peptide so that coupling occurredvia its thiol group to maleimide activated KLH protein.

TABLE 55 Description of the four selected peptides Localisation on humanLAMP1 sequence of SEQ ID NO: 24 Immunogen SEQ ID 47-61 Peptide 1-KLH SEQID NO: 90 140-155 Peptide 2-KLH SEQ ID NO: 91 307-321 Peptide 3-KLH SEQID NO: 92 360-375 Peptide 4-KLH SEQ ID NO: 82

Immunisation and Obtention of Polyclonal Antibodies.

A total of three programs of rabbit immunisations were performed.Rabbits were immunized in the first program, with peptide 1 SEQ ID NO:90 and peptide 2 of SEQ ID NO: 91, in the second program with peptide 4of SEQ ID NO: 82 and peptide 3 of SEQ IOD NO: 92 and in the thirdprogram with heated denatured human LAMP1::histag protein produced asdescribed in example 6.2. In brief, the immunisation schedule comprisedfour injections and a final bleed after 28 days. Polyclonal response wasdetermined by ELISA on a sample from the final bleeds.

Purifications of Polyclonal Antibodies.

Reactive AF-aminoTOYOPEARL was used to couple each peptide described onTable 55 and to generate four affinity chromatography columns. The serumfinal bleeds on rabbits immunized with the respective peptides werepurified by peptide affinity chromatography. The purified polyclonalbatches were then characterized by SDS-PAGE and ELISA.

The serum final bleed on the rabbit immunized with LAMP1 protein waspurified by protein G affinity chromatography. The purified polyclonalbatch was then characterized by SDS-PAGE.

Example 17.1.2: IHC Screening and Identification of Polyclonal rAb4(Rabbit Antibody 4) Obtained by Peptide 4 Immunization

Rabbit polyclonal antibodies generated with peptides described inexample 17.1.1 were tested by IHC on FFPE sample of colon adenocarcinomapatient derived xenograft CR-LRB-010P and human breast carcinoma. Afterantigen retrieval procedure and endogen biotins blocking steps, slideswere incubated with the primary anti-antibody for 1 hour at 24° C.Negative controls were performed by omission of the primary antibody.The biotin free anti-rabbit UltraMap™ horseradish peroxidase (HRP)conjugate (760-4315, Ventana Medical Systems, Inc, USA) was used assecondary antibody system according to manufacturer's recommendations.Negative controls were performed by omission of the primary antibody. Acounterstaining step was done with hematoxylin (760-2037, VentanaMedical Systems, Inc, USA) and bluing reagent was applied (760-2037,Ventana Medical Systems, Inc, USA). Stained slides were dehydrated andcoverslipped with cytoseal XYL (8312-4, Richard-Allan Scientific, USA).Only antibodies from peptide 4 of SEQ ID NO: 82 immunization displayedLAMP1 membrane reinforcement in FFPE samples as shown in FIG. 38.

Example 17.1.3: Validation of Polyclonal Rabbit (rAb4) Batch

ICC with Cells Expressing or not LAMP1 at the Membrane

Human-LAMP1 and empty-vector HEK transfected cells were tested with thepolyclonal rabbit rAb4 Antibody by immunocytochemistry (ICC) in FFPEformat. High level of intracellular and surface cell LAMP1immunostaining was obtained using the polyclonal rabbit rAb4 Antibody Abat 1 μg/mL as shown in FIG. 39.

Affinity to LAMP1 Protein

Secreted LAMP1::histag (29-382) with SEQ ID NO: 28 described in example6.2 was used to determine the affinity of the polyclonal antibodies polyrAb4 by ELISA as described in example 6.3. The polyclonal rabbitantibody poly rAb4 binds to LAMP1 with an EC₅₀ of around 3 nM whereasMAb1 binds with an EC₅₀ of 0.16 nM.

Example 17.2: Obtention and Characterization of Mouse MonoclonalAntibodies that LED to LAMP1 Membrane Reinforcement on FFPE TumorTissues Example 171.1: Mouse Immunisation and Selection of Mature IgGLAMP1-Secreting Hybridoma

While immunizations have been performed with diverse protein antigensincluding recombinant chimer human/mouse LAMP1 protein, recombinantdenatured human LAMP1 protein or recombinant human LAMP1 protein, theseapproaches were not successful in identifying antibody able to detectLAMP1 membrane reinforcement on FFPE tumor tissues. These approachesused immunization protocol described in example 2 for generation ofanti-LAMP1 monoclonal antibodies and LAMP1 proteins described in example6.2. On the contrary, the peptide 4-based immunization strategy has beenshown to identify an antibody eligible to detect LAMP1 membranereinforcement on FFPE tumor tissues.

Therefore mouse were immunised with peptide 4 and anti-LAMP1-secretinghybridomas were selected as described below.

Generation of Anti-LAMP1 Monoclonal Antibodies

Five BALB/cJ mice, about 6-8 weeks old (Charles River) were immunizedwith 40 μg of peptide 4 of SEQ ID NO: 82 using RIMMS approach asdescribed by Kilpatrick et al.; hybridoma, 1997: volume 16, number 4. Bcells immortalization using P3X63-AG8.653 (ATCC, ref CRL-1580) as fusionpartner and hybridoma selection was performed as described in example 2.

Selection of Anti-LAMP1 Antibodies by ELISA

The primary screen was an enzyme-linked immunosorbent assay (ELISA)assay (described in example 6.3 for anti-LAMP1 IgG production) using theLAMP1::histag protein described in example 6.2 as capturing antigen.

Example 17.2.2: IHC Screening and Identification of MAb4

As the same manner as in example 17.1.2, IHC screening was performedwith the hybridoma supernatant to identify mouse monoclonal antibodyshowing LAMP1 membrane reinforcement on FFPE sample of colonadenocarcinoma patient derived xenograft CR-LRB-010P. The biotin freeanti-mouse UltraMap™ horseradish peroxidase (HRP) conjugate (760-152,Ventana Medical Systems, Inc, USA) was used as secondary antibody systemaccording to manufacturer's recommendations.

The supernatant of the selected hybridoma 88LAMP1-2 displayed membranereinforcement immunostaining in FFPE sample of colon adenocarcinomapatient derived xenograft CR-LRB-010P. Other irrelevant antibodies werenegative or displayed intracellular immunostaining as shown in FIG. 40.

Example 17.2.3: Validation of Hybridoma 88LAMP1-2

Purification and Characterisation of Mab4 Obtained from Hybridoma88LAMP1-2

Hybridoma 88LAMP1-2 was produced in medium A Clonacell-Hy (StemCellTechnologies #03801) supplemented with 5% HCS (PAA; #F05-009) at the 400mL scale and purified by protein A affinity chromatography. The purifiedantibody MAb4 was characterized by SDS-PAGE, and Mass Spectrometry.Masses of heavy and light chains from MAb4 were identified as reportedin example 7 and are reported on the Table 55 below. Nucleic acidsequences encoding the variable domains were retrieved from hybridomacells by RT-PCR as described in example 7. The corresponding amino acidsfrom the heavy and light chains led to masses in agreement with therespective masses from MAb4.

TABLE 56 Mass characterization of MAb4 Mass obtained by Mass calculatedfrom mAb4 Isotype Mass Spectrometry amino acid sequence Heavy chainmlgG1 (G0F) 50 169 Da 50 168 Da Light chain mCk 23651 Da 23 650 Da

Example 17.3: In Vitro Characterisation of MAb4 Example 17.3.1: ApparentAffinity to Human LAMP1 and Cynomolgus LAMP1 by ELISA

Antibody MAb4 was assessed for its ability to bind primate LAMP1 proteinby enzyme-linked immunosorbent assay (ELISA) assay as described inexample 4.7 and EC50 values determined as described in example 6.2.Antibody MAb4 binds to human LAMP1 and cynomolgus LAMP1 with similaraffinity in range of 0.2 to 0.4 nM as shown in Table 57 below.

TABLE 57 EC₅₀ determined by ELISA values on recombinant human LAMP1 andcynomolgus LAMP1 LAMP1 protein EC₅₀ Human LAMP1 0.39 nM cynomolgus LAMP10.22 nM

Example 17.3.2: Specificity to LAMP1

LAMP2 is the closest member of the LAMP family with 35% sequenceidentity to LAMP1. Specificity of MAb4 was evaluated by ELISA asdescribed in example 4.6 with either LAMP1 or LAMP2 soluble proteins, asshown in FIG. 41. No binding to LAMP2 was detected with MAb4 and adifference of more than 100 fold is observed between the EC₅₀ of MAb4towards LAMP1 versus LAMP2.

Example 17.3.3: Binding of Antibody MAb4 to Multiple Cancer Cells andDetermination of Antibody Binding Capacity by Flow Cytometry

Antibody MAb4 was found to be able of binding to multiple tumor cells byFlow Cytometry using the conditions described in example 4.1. The panelof tumor cells comprises Patient-derived tumor xenografts from differentorigins and tumor cell lines. The Mean Flurescence Intensity (MFI)obtained from the flow cytometry analysis is reported in Table 58. Table59 summarizes the antibody binding capacity results.

TABLE 58 Mean Florescence Intensity by FACS on Patient-derivedxenografts Mean Florescence Intensity (MFI) CR-IGR-034P/colorectal 424LUN-NIC-006/lung 162 LUN-NIC-033/lung 154 BRE-IGR-0159/breast 400Colo205/colon 7

TABLE 59 Antibody Binding Capacity by FACS on Patient-derived xenograftAntibody Binding Capacity (ABC) MAb4 PDX/origin CR-IGR-034P/colorectal260 000  LUN-NIC-006/lung 92 000 LUN-NIC-033/lung 87 000 Celllines/origin Colo205/colon   3000

Example 17.3. 4: Apparent Affinity of Antibody MAb4 to Human PrimaryColon Tumor PDX (CR-IGR-034P) by Row Cytometry

Apparent affinity of antibody MAb4 was evaluated to human primary colontumor PDX CR-IGR-034P by Flow Cytometry using the conditions describedin example 4.1. EC₅₀ obtained with CR-IGR-034P with MAb4 was 1.3 nM.

Example 17.3. 5: IHC Validation

Results obtained with purified batch are similar to those obtained inexample 17.2.2 with none purified MAb4.

1. An immunoconjugate comprising a) an antibody or antigen bindingfragment thereof wherein said antibody or antigen binding fragmentthereof i) binds to a first lumenal domain of human LAMP1 protein, orii) binds to a domain consisting of loops one to three of human LAMP1protein, or iii) binds to a fourth loop of human LAMP1 protein; and b)at least one growth inhibitory agent, wherein the at least one growthinhibitory agent is linked to the antibody or antigen binding fragmentthereof. 2-5. (canceled)
 6. The immunoconjugate according to claim 1,wherein the antibody or antigen binding fragment thereof competes forbinding to the first lumenal domain of human LAMP1 protein with anantibody selected from the group consisting of (i) an antibodycomprising a heavy chain variable domain having an amino acid sequenceof SEQ ID NO: 1 and a light chain variable domain having an amino acidsequence SEQ ID NO: 5; or (ii) an antibody comprising a heavy chainvariable domain having an amino acid sequence of SEQ ID NO: 8 and alight chain variable domain having an amino acid sequence SEQ ID NO: 12;or (iii) an antibody comprising a heavy chain variable domain having anamino acid sequence of SEQ ID NO: 15 and a light chain variable domainhaving an amino acid sequence of SEQ ID NO: 16; or (i) an antibodycomprising a heavy chain variable domain having an amino acid sequenceof SEQ ID NO: 42 and a light chain variable domain having an amino acidsequence of SEQ ID NO: 46; or (iv) an antibody comprising a heavy chainvariable domain having an amino acid sequence of SEQ ID NO: 42 and alight chain variable domain having an amino acid sequence of SEQ ID NO:51; or (v) an antibody comprising a heavy chain variable domain havingan amino acid sequence of SEQ ID NO: 53 and a light chain variabledomain having an amino acid sequence of SEQ ID NO: 56; or (vi) anantibody comprising a heavy chain variable domain having an amino acidsequence of SEQ ID NO: 54 and a light chain variable domain having anamino acid sequence of SEQ ID NO: 57; or (vii) an antibody comprising aheavy chain variable domain having an amino acid sequence of SEQ ID NO:55 and a light chain variable domain having an amino acid sequence ofSEQ ID NO:
 58. 7-8. (canceled)
 9. The immunoconjugate according to claim1, wherein the antibody or antigen binding fragment thereof comprises:(i) a CDR1-H having an amino acid sequence of SEQ ID NO: 2, a CDR2-Hhaving an amino acid sequence of SEQ ID NO: 3, a CDR3-H having an aminoacid sequence of SEQ ID NO: 4, a CDR1-L having an amino acid sequence ofSEQ ID NO: 6, a CDR2-L having an amino acid sequence of DTS, and aCDR3-L having an amino acid sequence of SEQ ID NO: 7; or (ii) a CDR1-Hhaving an amino acid sequence of SEQ ID NO: 9, a CDR2-H having an aminoacid sequence of SEQ ID NO: 10, a CDR3-H having an amino acid sequenceof SEQ ID NO: 11, a CDR1-L having an amino acid sequence of SEQ ID NO:13, a CDR2-L having an amino acid sequence of AAS, and a CDR3-L havingan amino acid sequence of SEQ ID NO: 14; or (iii) a CDR1-H having anamino acid sequence of SEQ ID NO: 43, a CDR2-H having an amino acidsequence of SEQ ID NO: 44, a CDR3-H having an amino acid sequence of SEQID NO: 45, a CDR1-L having an amino acid sequence of SEQ ID NO: 47, aCDR2-L having an amino acid sequence of YTS, and a CDR3-L having anamino acid sequence of SEQ ID NO: 48 or SEQ ID NO:
 52. 10-12. (canceled)13. The immunoconjugate according to claim 1 wherein the antibody is achimeric or a humanised antibody.
 14. The immunoconjugate according toclaim 1, wherein the antibody comprises: i) a heavy chain having anamino acid sequence of SEQ ID NO: 17 and a light chain having an aminoacid sequence of SEQ ID NO: 18; or ii) a heavy chain having an aminoacid sequence of SEQ ID NO: 19 and a light chain having an amino acidsequence of SEQ ID NO: 20; or iii) a heavy chain having an amino acidsequence of SEQ ID NO: 21 and a light chain having an amino acidsequence of SEQ ID NO: 22; or iv) a heavy chain having an amino acidsequence of SEQ ID NO: 49 and a light chain having an amino acidsequence of SEQ ID NO: 50, or v) a heavy chain having an amino acidsequence of SEQ ID NO: 49 and a light chain having an amino acidsequence of SEQ ID NO: 81, or vi) a heavy chain having an amino acidsequence of SEQ ID NO: 60 and a light chain having an amino acidsequence of SEQ ID NO: 59; or vii) a heavy chain having an amino acidsequence of SEQ ID NO: 62 and a light chain having an amino acidsequence of SEQ ID NO: 61; or Viii) a heavy chain having an amino acidsequence of SEQ ID NO: 64 and a light chain having an amino acidsequence of SEQ ID NO:
 63. 15. (canceled)
 16. The immunoconjugateaccording to claim 1, wherein the antibody or antigen binding fragmentthereof binds to a region of Loop 4 comprising the amino acids 360 to375 of human LAMP1 (SEQ ID NO: 82).
 17. The immunoconjugate according toclaim 16, wherein the antibody or antigen binding fragment thereofcomprises a CDR1-H having an amino acid sequence of SEQ ID NO: 83, aCDR2-H having an amino acid sequence of SEQ ID NO: 84, a CDR3-H havingan amino acid sequence of SEQ ID NO: 85, a CDR1-L having an amino acidsequence of SEQ ID NO: 86, a CDR2-L having an amino acid sequence ofNAK, and a CDR3-L having an amino acid sequence of SEQ ID NO:
 87. 18-22.(canceled)
 23. The immunoconjugate according to claim 1, wherein said atleast one growth inhibitory agent is a cytotoxic agent or a radioactiveisotope. 24-26. (canceled)
 27. The immunoconjugate conjugate accordingto claim 23, wherein said growth inhibitory agent is(N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine) DM1 orN^(2′)-deacetyl-N^(2′)(4-methyl-4-mercapto-1-oxopentyl)-maytansine(DM4).
 28. (canceled)
 29. The immunoconjugate according to claim 1,wherein said growth inhibitory agent is covalently attached to theantibody or antigen binding fragment thereof via a linker is selectedfrom the group consisting of N-succinimidyl pyridyldithiobutyrate(SPDB), 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB),and succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).30. The immunoconjugate according to claim 29, wherein said linker isN-succinimidyl pyridyldithiobutyrate (SPDB) and the growth inhibitoryagent isN^(2′)-deacetyl-N^(2′)-(4-methyl-4-mercapto-1-oxopentyl)-maytansine(DM4).
 31. The immunoconjugate according to claim 29, wherein saidlinker is 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB)and the growth inhibitory agent isN^(2′)-deacetyl-N^(2′)-(4-methyl-4-mercapto-1-oxopentyl)-maytansine(DM4).
 32. The immunoconjugate according to claim 29, wherein saidlinker is succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate(SMCC) and the growth inhibitory agent isN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1). 33.The immunoconjugate according to claim 27, wherein the immunoconjugateis characterised by a drug-to-antibody ratio (DAR) ranging from 1 to 10,the DAR being calculated from the ratio of the drug concentration tothat of the antibody:DAR=C _(D) /C _(A)whereinC _(D)=[(ε_(A280) ×A ₂₅₂)−(ε_(A252) ×A₂₈₀)]/[(ε_(D252)×ε_(A280))−(ε_(A252)×ε_(D280))]C _(A) =[A ₂₈₀−(C _(D)×ε_(D280))]/ε_(A280) and ε_(D252) and ε_(D280) arerespectively the molar extinction coefficients of the drug at 252 nm and280 nm; ε_(A252) and ε_(A280) are respectively the molar extinctioncoefficients of the antibody at 252 nm and 280 nm; (A₂₅₂) and A₂₈₀ arerespectively the absorbances for the conjugate at 252 nm (A₂₅₂) and at280 nm (A₂₈₀), measured using a classic spectrophotometer apparatus. 34.An isolated antibody or antigen binding fragment thereof, wherein saidantibody or antigen binding fragment thereof binds to i) a first lumenaldomain of human LAMP1 protein, or ii) binds to a domain consisting ofloops one to three of human LAMP1 protein, or iii) binds to a fourthloop of human LAMP1 protein.
 35. The isolated antibody or antigenbinding fragment thereof according to claim 34, wherein the antibody orantigen binding fragment thereof competes for binding to the firstlumenal domain of human LAMP1 protein with an antibody selected from thegroup consisting of (i) an antibody comprising a heavy chain variabledomain having an amino acid sequence of SEQ ID NO: 1 and a light chainvariable domain having an amino acid sequence of SEQ ID NO: 5; or (ii)an antibody comprising a heavy chain variable domain having an aminoacid sequence of SEQ ID NO: 8 and a light chain variable domain havingan amino acid sequence of SEQ ID NO: 12; or (iii) an antibody comprisinga heavy chain variable domain having an amino acid sequence of SEQ IDNO: 15 and a light chain variable domain having an amino acid sequenceof SEQ ID NO: 16; or (ii) an antibody comprising a heavy chain variabledomain having an amino acid sequence of SEQ ID NO: 42 and a light chainvariable domain having an amino acid sequence of SEQ ID NO: 46; or (iv)an antibody comprising a heavy chain variable domain having an aminoacid sequence of SEQ ID NO: 42 and a light chain variable domain havingan amino acid sequence of SEQ ID NO: 51; or (v) an antibody comprising aheavy chain variable domain having an amino acid sequence of SEQ ID NO:53 and a light chain variable domain having an amino acid sequence ofSEQ ID NO: 56, or (vi) an antibody comprising a heavy chain variabledomain having an amino acid sequence of SEQ ID NO: 54 and a light chainvariable domain having an amino acid sequence of SEQ ID NO: 57, or (vii)an antibody comprising a heavy chain variable domain having an aminoacid sequence of SEQ ID NO: 55 and a light chain variable domain havingan amino acid sequence of SEQ ID NO:
 58. 36-41. (canceled)
 42. Anisolated anti-LAMP-1 antibody or antigen binding fragment thereofaccording to claim 34, wherein said antibody or antigen binding fragmentthereof comprises: (i) a CDR1-H having an amino acid sequence of SEQ IDNO: 2, a CDR2-H having an amino acid sequence of SEQ ID NO: 3, and aCDR3-H having an amino acid sequence of SEQ ID NO: 4; and a CDR1-Lhaving an amino acid sequence of SEQ ID NO: 6, a CDR2-L having an aminoacid sequence of DTS, and a CDR3-L having an amino acid sequence of SEQID NO: 7; or (ii) a CDR1-H having an amino acid sequence of SEQ ID NO:9, a CDR2-H having an amino acid sequence of SEQ ID NO: 10, a CDR3-Hhaving an amino acid sequence of SEQ ID NO: 11; and a CDR1-L having anamino acid sequence of SEQ ID NO: 13, a CDR2-L having an amino acidsequence of AAS, and a CDR3-L having an amino acid sequence of SEQ IDNO: 14; or (iii) a CDR1-H having an amino acid sequence of SEQ ID NO:43, a CDR2-H having an amino acid sequence of SEQ ID NO: 44, and aCDR3-H having an amino acid sequence of SEQ ID NO: 45, and a CDR1-Lhaving an amino acid sequence of SEQ ID NO: 47, a CDR2-L having an aminoacid sequence of YTS, and a CDR3-L having an amino acid sequence of SEQID NO: 48 or SEQ ID NO: 52; or (iv) CDR1-H having an amino acid sequenceof SEQ ID NO: 83, a CDR2-H having an amino acid sequence of SEQ ID NO:84, a CDR3-H having an amino acid sequence of SEQ ID NO: 85, and aCDR1-L having an amino acid sequence of SEQ ID NO: 86, a CDR2-L havingan amino acid sequence of NAK, and a CDR3-L having an amino acidsequence of SEQ ID NO:
 87. 43. (canceled)
 44. A pharmaceuticalcomposition comprising an immunoconjugate according to claim 1 and apharmaceutically acceptable carrier. 45-50. (canceled)
 51. An isolatednucleic acid comprising a sequence encoding an antibody or antigenbinding fragment thereof wherein said antibody or antigen bindingfragment thereof comprises (i) a CDR1-H having an amino acid sequence ofSEQ ID NO: 2, a CDR2-H having an amino acid sequence of SEQ ID NO: 3,and a CDR3-H having an amino acid sequence of SEQ ID NO: 4; and a CDR1-Lhaving an amino acid sequence of SEQ ID NO: 6, a CDR2-L having an aminoacid sequence of DTS, and a CDR3-L having an amino acid sequence of SEQID NO: 7; or (ii) a CDR1-H having an amino acid sequence of SEQ ID NO:9, a CDR2-H having an amino acid sequence of SEQ ID NO: 10, a CDR3-Hhaving an amino acid sequence of SEQ ID NO: 11; and a CDR1-L having anamino acid sequence of SEQ ID NO: 13, a CDR2-L having an amino acidsequence of AAS, and a CDR3-L having an amino acid sequence of SEQ IDNO: 14; or (iii) a CDR1-H having an amino acid sequence of SEQ ID NO:43, a CDR2-H having an amino acid sequence of SEQ ID NO: 44, and aCDR3-H having an amino acid sequence of SEQ ID NO: 45, and a CDR1-Lhaving an amino acid sequence of SEQ ID NO: 47, a CDR2-L having an aminoacid sequence of YTS, and a CDR3-L having an amino acid sequence of SEQID NO: 48 or SEQ ID NO: 52; or (iv) CDR1-H having an amino acid sequenceof SEQ ID NO: 83, a CDR2-H having an amino acid sequence of SEQ ID NO:84, a CDR3-H having an amino acid sequence of SEQ ID NO: 85, and aCDR1-L having an amino acid sequence of SEQ ID NO: 86, a CDR2-L havingan amino acid sequence of NAK, and a CDR3-L having an amino acidsequence of SEQ ID NO:
 87. 52. A host cell which has been transformed bya nucleic acid according to claim
 51. 53. An in vitro method ofselecting patients with cancer who are likely to respond to anti-LAMP1therapy, wherein said method comprises: a) determining, in a biologicalsample of a patient with cancer which includes cancer cells, if saidpatient harbors a LAMP1 gene copy number gain; and b) selecting thepatient based on the presence of LAMP1 gene copy number gain, andwherein said patient is selected as likely to respond to anti-LAMP1therapy if said patient harbors a LAMP1 gene copy number gain. 54-68.(canceled)
 69. A method a patient in need thereof comprisingadministering to the patient an antibody or antigen binding fragmentthereof wherein said antibody or antigen binding fragment thereof i)binds to the first lumenal domain of human LAMP1 protein, or binds to adomain consisting of loops one to three of human LAMP1 protein, or HDbinds to a fourth loop of human LAMP1 protein; wherein the patient has aLAMP1 expressing cancer.
 70. The method according to claim 69, whereinthe antibody or antigen binding fragment thereof is linked to at leastone growth inhibitory agent, wherein the at least one growth inhibitory.